Fibronectin type iii domain-based multimeric scaffolds

ABSTRACT

The present invention provides fibronectin type III (Fn3)-based multimeric scaffolds that specifically bind to one or more specific target antigen. The invention further provides bispecific Fn3-derived binding molecules that bind to two or more target antigens simultaneously, fusions, conjugates, and methods to increase the stability of Fn3-based binding molecules. Furthermore, the present invention is related to a prophylactic, therapeutic or diagnostic agent, which contains Fn3-based multimeric scaffolds.

REFERENCE TO THE SEQUENCE LISTING

This application incorporates by reference a Sequence Listing submitted with this application via EFS-Web as text file entitled “2943.011PC01_sequence_listing.txt” created on Apr. 12, 2011 and having a size of 221 kilobytes.

FIELD OF THE INVENTION

The present invention relates in general to the field of antibody mimetics, specifically to multimeric scaffolds based on the fibronectin type III (Fn3) domain useful, for example, for the generation of products having novel binding characteristics.

BACKGROUND

Biomolecules capable of specific binding to a desired target epitope are of great importance as therapeutics, research, and medical diagnostic tools. A well known example of this class of molecules is the antibody. Antibodies can be selected that bind specifically and with affinity to almost any structural epitope. However, classical antibodies are structurally complex heterotetrameric molecules with are difficult to express in simple eukaryotic systems. As a result, most antibodies are produced using complex and expensive mammalian cell expression systems.

Proteins having relatively defined three-dimensional structures, commonly referred to as protein scaffolds, may be used as reagents for the design of engineered products. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomization is often carried out to produce libraries of proteins from which desired products may be selected.

One particular area in which such scaffolds are useful is the field of antibody mimetic design. Antibody mimetics, i.e., small, non-antibody protein therapeutics, capitalize on the advantages of antibodies and antibody fragments, such as high affinity binding of targets and low immunogenicity and toxicity, while avoiding some of the shortfalls, such as the tendency for antibody fragments to aggregate and be less stable than full-length IgGs.

These drawbacks can be addressed by using antibody fragments created by the removal of parts of the antibody native fold. However, this often causes aggregation when amino acid residues which would normally be buried in a hydrophobic environment such as an interface between variable and constant domain become exposed to the solvent.

One example of an scaffold-based antibody mimetic is based on the structure of a fibronectin module of type III (FnIII), a domain found widely across phyla and protein classes, such as in mammalian blood and structural proteins. The FnIII domain occurs often in various proteins, including fibronectins, tenascin, intracellular cytoskeletal proteins, cytokine receptors and prokaryotic enzymes (Bork and Doolittle, Proc. Natl. Acad. Sci. USA 89:8990-8894, 1992; Bork et al., Nature Biotechnol. 15:553-557, 1997; Meinke et al., J. Bacteriol. 175:1910-1918, 1993; Watanabe et al., J. Biol. Chem. 265:15659-15665, 1990). PCT Publication No: WO 2009/058379 describes scaffolds based on the FnIII domain, in particular, the third FnIII domain of human tenascin C. FnIII domains comprise seven beta strands, designated N-terminus to C-terminus A, B, C, D, E, F, and G strands, each strand separated by a loop region wherein the loop regions are designated N-terminus to C-terminus, AB, BC, CD, DE, EF, and FG loops. Although the FnIII domain is not an immunoglobulin, the overall fold of the third FnIII domain of human tenascin C domain is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain, which comprises the entire antigen recognition unit in camel and llama IgG. This makes it possible to display the three fibronectin loops on each opposite side of a FnIII domain, e.g., the third FnIII domain of human tenascin C in relative orientations similar to those of CDRs in native antibodies.

There is a need to develop improved stable, artificial antibody-like molecules, having increased specificity, affinity, avidity, and stability for a variety of therapeutic and diagnostic applications, as well as screening methods for identifying such molecules.

Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.

SUMMARY OF THE INVENTION

The invention provides recombinant multimeric scaffold comprising two fibronectin type III (FnIII) monomer scaffolds derived from one or more FnIII domains of interest (FOI), wherein (a) each FnIII monomer scaffold comprises a plurality of beta strands linked to a plurality of loop regions, (b) the FnIII monomer scaffolds are connected in tandem, wherein at least one of the monomers comprises a non-naturally occurring intramolecular disulfide bond, (c) the recombinant multimeric scaffold specifically binds to at least one target, and (d) the action on the target is improved over that of a cognate FnIII monomer scaffold.

The invention also provides recombinant multimeric scaffold comprising 3 fibronectin type III (FnIII) monomer scaffolds derived from one or more FnIII domains of interest (FOI) wherein (a) each FnIII monomer scaffold comprises a plurality of beta strands linked to a plurality of loop regions, (b) the recombinant multimeric FnIII scaffold specifically binds to at least one target, and (c) the action on the target is improved over that of a cognate FnIII monomer scaffold.

In some embodiments, the multimeric scaffolds of the invention comprise 3, 4, 5, 6, 7, or 8 FnIII monomer scaffolds. In some embodiments, all of the FnIII monomer scaffolds in the multimeric scaffold are in tandem. In other embodiments, at least two FnIII monomer scaffolds in a multimeric scaffold comprise a non-naturally occurring intramolecular disulfide bond. In some other embodiments, the multimeric scaffold of the invention binds to at least 2 targets. In some embodiments, at least one FnIII monomer scaffold in a multimeric scaffold is connected directly, by a linker, or by a heterologous moiety to 2, 3, 4, 5, or 6 other FnIII monomer scaffolds. In some embodiments, the multimeric scaffold of the invention comprises 7, 8, 9, 10, 11 or 12 FnIII monomer scaffolds, which in some embodiments can all be in tandem.

In some embodiments, at least two FnIII monomer scaffolds in a multimeric scaffold are connected by a linker. In other embodiments, at least two FnIII monomer scaffolds in a multimeric scaffold are directly connected without a linker interposed between the FnIII monomer scaffolds. In some embodiments, the plurality of beta strands in at least one FnIII monomer scaffold in the multimeric scaffold comprises seven beta strands designated A, B, C, D, E, F, and G. In other embodiments, the plurality of loop regions in at least one FnIII monomer scaffold in the multimeric scaffold comprises six loop regions designated AB, BC, CD, DE, EF, and FG.

In some embodiments, for at least one FnIII monomer scaffold in a multimeric scaffold of the invention there is an improvement in binding over that of a cognate FnIII monomer scaffold wherein the improvement is in binding affinity and/or avidity.

In some embodiments, binding affinity for the target and protein stability are improved in the multimeric scaffold over those of a cognate FnIII monomer scaffold. In other embodiments, the binding avidity for the target and the protein stability of a multimeric scaffold are improved over those of a cognate FnIII monomer scaffold.

In some embodiments, at least one FnIII monomer scaffold in a multimeric scaffold of the invention comprises at least two non-naturally occurring intramolecular disulfide bonds. In some embodiments, the multimeric scaffold comprises a peptide linker. The peptide linker can be a flexible peptide linker. In some embodiments, the linker comprises a functional moiety, which is some cases can be an immunoglobulin or a fragment thereof.

In some embodiments, at least one of the FnIII monomer scaffolds in a multimeric scaffold is fused to a heterologous moiety, such as a protein, a peptide, a protein domain, a linker, a drug, a toxin, a cytotoxic agent, an imaging agent, a radionuclide, a radioactive compound, an organic polymer, an inorganic polymer, a polyethylene glycol (PEG), biotin, a human serum albumin (HSA), a HSA FcRn binding portion, an antibody, a domain of an antibody, an antibody fragment, a single chain antibody, a domain antibody, an albumin binding domain, an enzyme, a ligand, a receptor, a binding peptide, a non-FnIII scaffold, an epitope tag, a recombinant polypeptide polymer, a cytokine, and a combination of two or more of said moieties.

In some embodiments, more than two of the FnIII monomer scaffolds in a multimeric scaffold are connected by linkers, and at least one linker is structurally and/or functionally different from the other linkers. In other embodiments, the FnIII monomer scaffolds in a multimeric FnIII scaffold are connected in a branched format. In other embodiments, some FnIII monomer scaffolds in the multimeric scaffold are connected in a linear tandem format and some FnIII monomer scaffolds are connected in a branched format.

In some embodiments, at least two FnIII monomer scaffolds in the multimeric scaffold are identical, whereas is some other embodiments at least two FnIII monomer scaffolds are different. In some embodiments, the multimeric scaffold is a receptor agonist. In other embodiments, the multimeric scaffold is a receptor antagonist.

In some embodiments, at least two FnIII monomer scaffolds in the multimeric scaffold bind the same target at the same epitope. In other embodiments, at least two FnIII monomer scaffolds in a multimeric scaffold bind the same target at different epitopes. In some embodiments, the different epitopes are non-overlapping epitopes, whereas in other embodiments the different epitopes are overlapping epitopes.

In some embodiments, at least one FOI is selected from the group consisting of: an animal FnIII domain, a bacterial FnIII domain, an archaea FnIII domain, and a viral FnIII domain. This at least one FOI can comprise a sequence selected from the group consisting of any one of SEQ ID NOs: 1-34, 59, 69, and any of the sequences presented in FIG. 16. In some embodiments, the at least one FOI is an FnIII domain from a hyperthermophilic archaea.

In some embodiments, the FOI comprises the third FnIII domain of human tenascin C (SEQ ID NO: 4) or a functional fragment thereof. In some embodiments, the FOI comprises the 14th FnIII domain of human fibronectin (SEQ ID NO: 69) or a functional fragment thereof, or the 10th FnIII domain of human fibronectin (SEQ ID NO: 54) or a functional fragment thereof.

In some embodiments, the FOI for each FnIII monomer in a multimeric scaffold comprises the third FnIII domain of human tenascin C (SEQ ID NO: 4) or a functional fragment thereof. In some embodiments, the functional fragment of the third FnIII domain of human tenascin C is an N-terminal truncated form (SEQ ID NO: 14).

In some embodiments, the beta strands of at least one of the FnIII monomer scaffolds in a multimeric scaffold have at least 90% sequence identity to the cognate beta strands in SEQ ID NO: 4. In some embodiments, for at least one FnIII monomer scaffold in a multimeric scaffold, the A beta strand domain comprises SEQ ID NOs: 41 or 42, the B beta strand comprises SEQ ID NO: 43, the C beta strand comprises SEQ ID NO: 44 or 131, the D beta strand comprises SEQ ID NO: 46, the E beta strand comprises SEQ ID NO: 47, the F beta strand comprises SEQ ID NO: 48, and the G beta strand comprises SEQ ID NO: 52.

In some embodiments, for at least one FnIII monomer scaffold in a multimeric scaffold, the AB loop comprises SEQ ID NO: 35, the CD loop comprises SEQ ID NO: 37, and the EF loop comprises SEQ ID NO: 39. In other embodiments, for at least one FnIII monomer scaffold in a multimeric scaffold, the BC loop comprises SEQ ID NO: 36, the DE loop comprises SEQ ID NO: 38 and the FG loop comprises SEQ ID NO: 40.

In some embodiments, for at least one FnIII monomer scaffold in a multimeric scaffold, the AB loop comprises SEQ ID NO: 35, the BC loop comprises SEQ ID NO: 97, 98, 99, 100, or 101, the CD loop comprises SEQ ID NO: 37, the DE loop comprises SEQ ID NO: 38, 102, 103, 104, or 105, the EF loop comprises SEQ ID NO: 39, and the FG loop comprises SEQ ID NO:106, 107, 108, 109, 110, or 111.

In some embodiments, for at least one FnIII monomer scaffold in a multimeric scaffold, the A beta strand comprises SEQ ID NO: 41 or 42, the B beta strand comprises SEQ ID NO: 43, the C beta strand comprises SEQ ID NO: 44, 45, or 131, the D beta strand comprises SEQ ID NO: 46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO: 49, 50 or 51, and the G beta strand comprises SEQ ID NO: 52 or 53.

In some embodiments, the FOI of at least one FnIII monomer scaffold comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2 and SEQ ID NO: 3.

In some embodiments, at least one FnIII monomer scaffold in a multimeric scaffold comprises the amino acid sequence:

-   -   IEV(X_(AB))_(n)ALITW(X_(BC))_(n)CELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n)YEVSLIC(X_(FG))_(n)KE         TFTT     -   wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG)         represent the amino acid residues present in the AB, BC, CD, DE,         EF, and FG loops, respectively, wherein X₁ represents amino acid         residue A or T, and wherein the length of the loop n is an         integer between 2 and 26.

In some embodiments, at least one FnIII monomer scaffold in a multimeric scaffold comprises the amino acid sequence:

-   -   IEV(X_(AB))_(n)ALITW(X_(BC))_(n)CELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n)YCVSLIS(X_(FG))_(n)KE         CFTT     -   wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG)         represent the amino acid residues present in the AB, BC, CD, DE,         EF, and FG loops, respectively, wherein X₁ represents amino acid         residue A or T, and wherein the length of the loop n is an         integer between 2 and 26.

In some embodiments, at least one FnIII monomer scaffold in a multimeric scaffold comprises the amino acid sequence:

-   -   IEV(X_(AB))_(n)ALITW(X_(BC))_(n)CELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n)YCVSLIC(X_(FG))_(n)KE         CFTT     -   wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG)         represent the amino acid residues present in the AB, BC, CD, DE,         EF, and FG loops, respectively, wherein X₁ represents amino acid         residue A or T, and wherein the length of the loop n is an         integer between 2 and 26.

In some embodiments, at least one FnIII monomer scaffold in a multimeric scaffold comprises an AB loop comprising SEQ ID NO: 35, a CD loop comprising SEQ ID NO: 37, and an EF loop comprising SEQ ID NO: 39. In other embodiments, at least one FnIII monomer scaffold in a multimeric scaffold comprises a BC loop comprising SEQ ID NO: 36, a DE loop comprising SEQ ID NO: 38, and an FG loop comprising SEQ ID NO: 40.

In some embodiments, at least one FnIII monomer scaffold in a multimeric scaffold comprises at least one BC loop, DE, loop or FG loop variant. In some embodiments, the BC loop variant comprises SEQ ID NO: 97, 98, or 168. In other embodiments, the DE loop variant comprises SEQ ID NO: 102, or 103. In some other embodiments, the FG loop variant comprises SEQ ID NO: 106, 108, 109, 169, or 170. In some embodiments the BC loop, DE, loop or FG loop variant comprises the amino acid sequence of the respective BC, DE, or FG loop of SEQ ID NO: 178, 195, 196, 197, 198, 199, 200, 205, 206, 207, or 208.

In some embodiments, the increased protein stability of at least one FnIII monomer scaffold is measured by differential scanning calorimetry (DSC), circular dichroism (CD), polyacrylamide gel electrophoresis (PAGE), protease resistance, isothermal calorimetry (ITC), nuclear magnetic resonance (NMR), urea denaturation, or guanidine denaturation.

In some embodiments, at least one FnIII monomer scaffold in a multimeric scaffold is affinity matured.

The invention also provides a method for obtaining a recombinant multimeric scaffold comprising: expressing, fusing or conjugating 2 fibronectin type III (FnIII) monomer scaffolds derived from one or more wild-type FnIII domains of interest (FOI), wherein (a) each FnIII monomer scaffold comprises a plurality of beta strands linked to a plurality of loop regions, (b) the FnIII monomer scaffolds are connected in tandem, wherein at least one of the FnIII monomer scaffolds comprises one non-naturally occurring intramolecular disulfide bond, (c) the recombinant multimeric scaffold specifically binds to at least one target, and (d) the binding for the target is improved over that of a cognate FnIII monomer scaffold.

The invention also provides a method for obtaining a recombinant multimeric scaffold comprising: expressing, fusing or conjugating at least 3 fibronectin type III (FnIII) monomer scaffolds derived from one or more wild-type FnIII domains of interest (FOI) wherein (a) each FnIII monomer scaffold comprises a plurality of beta strands linked to a plurality of loop regions, (b) the recombinant multimeric FnIII scaffold specifically binds to at least one target, and (c) the binding for the target is improved over that of a cognate FnIII monomer scaffold.

In some embodiments, at least one of the FOIs used in the methods described above comprises a sequence selected from the group consisting of any one of SEQ ID NOs:1-34, 54, 69, and any of the sequences presented in FIG. 16.

The invention also provides a nucleic acid encoding any of the multimeric scaffolds described above. In some embodiments, a vector is operably linked to the nucleic acid. In other embodiments, a host cell can comprise the vector.

The invention also provides a method of producing a recombinant multimeric scaffold comprising culturing a host cell under conditions in which the multimeric scaffold encoded by the nucleic acid molecule is expressed.

In other embodiments, the scaffolds of the invention are combined with a pharmaceutically acceptable excipient to yield a pharmaceutical composition.

The invention also provides a method for treating a cancer, an autoimmune disorder, an inflammatory disorder, or an infection in a patient in need thereof comprising administering an effective amount of the composition of a pharmaceutical composition comprising a scaffold of the invention.

The invention also provides a method of detecting a protein in a sample comprising labeling a multimeric FnIII scaffold of the invention or a conjugate comprising a scaffold of the invention, contacting the labeled multimeric FnIII scaffold or conjugate with a sample, and detecting complex formation between the multimeric FnIII scaffold or conjugate with the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

FIG. 1 shows linear, antibody-like and fusion formats of multivalent Tn3 scaffolds. Multivalent Tn3 scaffolds contain two or more Tn3 modules attached by a spacer indicated by a black octagonal block, where the spacer can be, e.g., a linker.

FIG. 2 shows TRAIL R2-specific multivalent Tn3 scaffolds, designated as A2 to A9, which were generated according to the three different molecular formats shown in FIG. 1 with valencies (number of Tn3 modules) varying from 2 to 8.

FIG. 3 shows non reducing SDS-PAGE analysis of crude bacterial media (right gel) and affinity purified samples (left gel) corresponding to linear tandem constructs designated A1 to A5, with valencies varying from 1 to 8, expressed in E. coli.

FIG. 4 shows a competition ELISA measuring binding of monovalent (A1) and multivalent (A2, A3) Tn3 scaffolds to TRAIL R2.

FIG. 5. shows a flow cytometry histogram of the TRAIL R2-specific multivalent scaffold A9 binding to H2122 cells compared to a cognate control scaffold (B9) that does not bind TRAIL R2.

FIG. 6A shows the effect of valency on the specific killing of the TRAIL R2-expressing cell line H2122 by multivalent scaffolds.

FIG. 6B shows the specificity of H2122 tumor cell killing by TRAIL R2-specific multivalent scaffolds.

FIG. 7A shows the effect of molecular format on killing of H2122 cells by TRAIL R2-specific multivalent scaffolds comprising 4 Tn3 modules.

FIG. 7B shows the effect of molecular format on killing of H2122 cells by TRAIL R2-specific multivalent scaffolds comprising 8 Tn3 modules.

FIG. 8A shows the specific killing of colorectal adenocarcinoma cell line Colo205 cells expressing TRAIL R2 by linearly fused tetra-(A3) and octavalent (A5) TRAIL R2-specific Tn3 scaffolds.

FIG. 8B shows the specific killing of leukemic line Jurkat cells expressing TRAIL R2 by linearly fused tetra-(A3) and octavalent (A5) TRAIL R2-specific Tn3 scaffolds.

FIG. 9A shows the design of murine CD40L-specific tandem bivalent Tn3 scaffolds (M13 constructs).

FIG. 9B shows the SDS-PAGE analysis of a purified monovalent M13 construct (CD40L-specific Tn3 construct), or tandem bivalent scaffolds with linkers containing 1, 3, 5 or 7 Gly₄Ser units (denoted as GS) joining two M13 modules. Monovalent M13 construct was run in lane 2, Construct C1 in lanes 3 and 7, Construct C2 in lanes 4 and 8, construct C3 in lanes 5 and 9, and construct C4 in lanes 6 and 10. Samples were run either non-reduced conditions (lanes 2-6) or reduced conditions (lanes 7-10).

FIG. 9C shows the competitive inhibition of MuCD40L binding to Murine CD40 receptor immobilized on a biosensor chip by MuCD40L-specific monovalent (M13) or bivalent tandem scaffolds. The half maximal inhibitory concentration (IC₅₀) for the various constructs is indicated.

FIG. 9D shows the inhibitory effect of MuCD40L-specific monovalent (M13) Tn3, bivalent tandem scaffolds, or antibody MR1 (an anti-MuCD40L antibody) on MuCD40L-induced CD86 expression on B cells.

FIG. 10 shows the expression levels of soluble monovalent and TRAIL R2/CD40L-bispecific tandem bivalent Tn3 scaffold constructs recombinantly expressed in E. coli analyzed by SDS-PAGE of the bacterial culture media. Monovalent scaffolds, A1 and 79 are shown in lanes 2 and 3, respectively. Tandem scaffold constructs comprising A1 and 79, joined in tandem by a Gly₄Ser amino acid linker of increasing length (cognate to constructs C5, C6, C7 and C8) are shown in lanes 4-7. The expressed constructs are indicated on the stained gel by an asterisk.

FIG. 11A shows the binding of bispecific Tn3 scaffolds to TRAIL R2 assayed using capture ELISA.

FIG. 11B shows the binding of bispecific Tn3 scaffolds to Human CD40L assayed using capture ELISA.

FIG. 12 shows the simultaneous binding of bispecific tandem Tn3 scaffolds C5, C6, C7, and C8 to TRAIL R2 and CD40L assayed using an AlphaScreen™ assay.

FIG. 13 shows the stability of Tn3 scaffolds in the present of guanidine-HCl. C_(m) (midpoint value) for each tested scaffold is indicated.

FIG. 14 shows the thermostability of three different Tn3 scaffolds with different loop sequences, but the same length FG loop (nine amino acids) compared to the parental Tn3 scaffold which has a longer FG loop analyzed by differential scanning calorimetry (DSC).

FIG. 15 shows the increase in stability in the presence of guanidine-HCl of Tn3 scaffolds having a nine amino acid length FG loop (P1C01, A6, and 71) compared to the parental (WT) Tn3 scaffold.

FIG. 16 shows a multiple sequence alignment of 103 different FnIII scaffolds based on structural analysis. Each FnIII sequence corresponds to a different FnIII three dimensional structure, identified according to its respective Protein Data Bank (PDB) structure and chain (e.g., 1V5J_A, corresponds to the sequence of chain A in the 1V5J PDB structure). The entire sequence for each FnIII sequence is shown over four consecutive panels starting with the A strand, and ending with the G strand. The loop regions are indicated at the top of the alignments with a solid line. The sequences are displayed in groups with the AB loop of each group indicated on FIGS. 16A, 16E, 16I, and 16M; the BC and CD loops of each group are indicated on FIGS. 16B, 16F, 16J, and 16N; the DE and EF loops are indicated on FIGS. 16C, 16G, 16K, and 16O; and the FG loop is indicated on FIGS. 16D, 16H, 16L, and 16P.

FIG. 17A shows a schematic representation and expression of a trispecific/trivalent Tn3 scaffold. The D1-1E11-79 scaffold contains a Synagis®-binding domain (D1), followed by a TRAIL R2-Fc binding domain (1E11), and a C-terminal Tn3 domain specific for human CD40L (79). A flexible (Gly₄Ser)₃ linker separates each domain.

FIG. 17B shows a SDS-PAGE (4-12% Bis-Tris) gel of the expressed and purified D1-1E11-79 scaffold. The expected molecular weight of this construct is approximately 34,081 Daltons.

FIG. 18A shows the simultaneous binding of the trispecific/trivalent Tn3 scaffold D1-1E11-79 to huCD40L and TRAIL R2-Fc using AlphaScreen binding analysis. AlphaScreen signal (ASS) shown as a function of TrailR2-Fc concentration.

FIG. 18B shows the simultaneous binding of the trispecific/trivalent Tn3 scaffold D1-1E11-79 to huCD40L and Synagis® using AlphaScreen binding analysis. AlphaScreen signal (ASS) shown as a function of Synagis® concentration.

FIG. 19 shows the simultaneous binding of the trispecific/trivalent Tn3 scaffold D1-1E11-79 to TRAIL R2-Fc and Synagis® using ELISA.

FIG. 20 shows a sequence alignment of parental TRAIL R2 binding clone 1C12 and its affinity matured derivatives. The position of the engineered disulfide bond is highlighted, the arrow indicates the location of the one framework mutation, and changes in the loops that arise during affinity maturation are shown in highlighted blocks A, B, C, and D.

FIG. 21 shows a CellTiter-Glo cell viability assay of the 1C12 clone and its affinity matured derivatives.

FIG. 22 shows concentration of G6 tandems as a function of time in mouse serum.

FIG. 23A shows a sequence alignment corresponding to the engineered enhancement of cyno cross reactivity for clone F4. The common feature among all of these clones is a mutation from D to G two amino acids before the DE loop.

FIG. 23B shows ELISA measurements of the inhibition of binding of either human or cyno TRAIL R2-Fc to F4 mod 1 coated plates by F4 or F4 mod 1 monomer.

FIG. 24A shows a sequence alignment corresponding to germlining of the clone F4 mod 1, specifically a comparison of F4, F4 mod 1 and F4 mod 12 to the TN3 germline.

FIG. 24B shows ELISA measurements of the inhibition of binding of either human or cyno TRAIL-R2-Fc to F4 mod 1 coated plates by F4, F4 mod 1, or F4 mod 12 monomer.

FIG. 24C shows a Colo205 cell killing assay comparing G6 tandem 6 to F4 mod 12 tandem 6.

FIG. 24D shows a Colo205 cell killing assay comparing G6 tandem 8 to F4 mod 12 tandem 8.

FIG. 25 shows an HT29 cell killing assay comparing the activity of G6 tandem 8 to F4 mod 12 tandem 8 in the TRAIL resistant cell line HT29.

FIG. 26 shows a sequence alignment corresponding to the clones tested in Antitope EpiScreen Immunogenicity analyses. Differences with respect to clone F4 mod 12 are highlighted.

FIG. 27A shows SEC traces of non-SEC-purified G6 tandem 8.

FIG. 27B shows SEC traces of SEC-purified G6 tandem 8.

FIG. 28 shows changes in tumor volume in Colo205 colorectal cancer xenograft models in response to different doses of the Tn3 TRAIL R2 agonists G6 tandem 6 and G6 tandem 8.

FIG. 29 shows changes in body weight in Colo205 colorectal xenograft models in response to different doses of the Tn3 TRAIL R2 agonists G6 tandem 6 and G6 tandem 8.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions or process steps, as such can vary. It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. The terms “a” (or “an”), as well as the terms “one or more,” and “at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this invention.

Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided.

Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.

The term “epitope” as used herein refers to a protein determinant capable of binding to a scaffold of the invention. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

The terms “fibronectin type III (FnIII) domain,” “FnIII domain” refer to polypeptides homologous to the human fibronectin type III domain having at least 7 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing solvent exposed loops which connect the beta strands to each other. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands. In certain embodiments, an FnIII domain comprises 7 beta strands designated A, B, C, D, E, F, and G linked to six loop regions designated AB, BC, CD, DE, EF, and FG, wherein a loop region connects each beta strand. FIG. 16 provides the primary sequence locations for the beta strands and loops for numerous FnIII domains based on analysis of their three dimensional structures. It should be noted that alternative definitions of these regions are known in the art. However, for these FnIII domains, the definitions in FIG. 16 will be used herein unless the context clearly dictates otherwise except that it will be understood that the N-terminus of the A strand and/or the C-terminus of the G strand may be truncated. The terms “fibronectin type III (FnIII) domain” and “FnIII domain” also comprise protein domains recognized to contain the Interpro IPRO08957 fibronectin type III domain signature as determined using the InterProScan program, or recognized to contain the Pfam PF00041 fibronectin type III domain signature as determined using Pfam_scan, HMMER, or any other program known in the art capable of comparing a protein sequence to a Hidden Markov model describing an FnIII domain. In addition, the terms include functional fragments and engineered FnIII domains, e.g., core-engineered FnIII domains (see, e.g., Ng et al., Nanotechnology 19: 384023, 2008).

The terms “Fibronectin type III (FnIII) scaffold” or “FnIII scaffold” refers to a polypeptide comprising an FnIII domain, or functional fragment thereof, wherein at least one loop is a non-naturally occurring variant of a FnIII domain/scaffold of interest, and wherein said FnIII scaffold, or functional fragment thereof is capable of binding a target, wherein the term “binding” herein preferably relates to a specific binding. As used herein a “non-naturally occurring variant” can vary by deletion, substitution or addition by at least one amino acid from the cognate sequences in a starting protein sequence (e.g., an FnIII domain/scaffold of interest), which may be a native FnIII domain sequence or a previously identified FnIII scaffold sequence. In certain embodiments, the A beta strand is truncated, for example one or more N-terminal residues of the A beta strand can be absent. In certain embodiments, the G beta strand is truncated, for example one or more C-terminal residues of the G beta strand may be absent. In certain embodiments, an FnIII scaffold comprises a non-naturally occurring variant of one or more beta strands. In certain embodiments, the beta strands of the FnIII scaffold exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more sequence identity to the primary sequences of the cognate beta strands of any one of SEQ ID NOs: 1-34, 54, or 69 or to the primary sequences of the beta strands of any of the FnIII domains shown in FIG. 16; or to the beta strands of a protein domain recognized to contain the Interpro IPRO08957 fibronectin type III domain signature as determined using the InterProScan program, or recognized to contain the Pfam PF00041 fibronectin type III domain signature as determined using Pfam_scan, HMMER, or any other program capable of comparing a protein sequence to a Hidden Markov model.

The term “DNA” refers to a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides.

The term “fusion protein” refers to protein that includes (i) one or more scaffolds of the invention joined to (ii) a second, different protein (i.e., a “heterologous” protein).

The term “heterologous moiety” is used herein to indicate the addition of a composition to a scaffold of the invention wherein the composition is not normally part of an FnIII domain. Exemplary heterologous moieties include proteins, peptides, protein domains, linkers, drugs, toxins, imaging agents, radioactive compounds, organic and inorganic polymers, and any other compositions which might provide an activity that is not inherent in the FnIII domain itself, including, but are not limited to, polyethylene glycol (PEG), a cytotoxic agent, a radionuclide, imaging agent, biotin, a dimerization domain (e.g. leucine zipper domain), human serum albumin (HSA) or an FcRn binding portion thereof, a domain or fragment of an antibody (e.g., antibody variable domain, a CH1 domain, a Ckappa domain, a Clambda domain, a CH2, or a CH3 domain), a single chain antibody, a domain antibody, an albumin binding domain, an IgG molecule, an enzyme, a ligand, a receptor, a binding peptide, a non-FnIII scaffold, an epitope tag, a recombinant polypeptide polymer, a cytokine, and the like.

The term “linker” as used herein refers to any molecular assembly that joins or connects two or more scaffolds. The linker can be a molecule whose function is to act as a “spacer” between modules in a scaffold, or it can also be a molecule with additional function (i.e., a “functional moiety’). A molecule included in the definition of “heterologous moiety” can also function as a linker.

The terms “linked” and “fused” are used interchangeably. These terms refer to the joining together of two or more scaffolds, heterologous moieties, or linkers by whatever means including chemical conjugation or recombinant means.

The terms “multimer,” “multimeric scaffold” and “multivalent scaffold” refer to a molecule that comprises at least two FnIII scaffolds in association. The scaffolds forming a multimeric scaffold can be linked through a linker that permits each scaffold to function independently. “Multimeric” and “multivalent” can be used interchangeably herein. A multivalent scaffold can be monospecific or bispecific.

The terms “domain” or “protein domain” refer to a region of a protein that can fold into a stable three-dimensional structure, often independently of the rest of the protein, and which can be endowed with a particular function. This structure maintains a specific function associated with the domain's function within the original protein, e.g., enzymatic activity, creation of a recognition motif for another molecule, or to provide necessary structural components for a protein to exist in a particular environment of proteins. Both within a protein family and within related protein superfamilies, protein domains can be evolutionarily conserved regions. When describing the component of a multimeric scaffold, the terms “domain,” “monomeric scaffold,” and “module” can be used interchangeably. By “native FnIII domain” is meant any non-recombinant FnIII domain that is encoded by a living organism.

The term ““sequence homology”” in relation to protein sequences refers to the similarity between two or more protein sequences, i.e., the percentage of amino acid residues that are either identical or conservative amino acid substitutions.

The terms “Percent (%) sequence similarity” and “Percent (%) homology” as used herein are considered equivalent and are defined as the percentage of amino acid residues in a candidate sequence that are identical with or conservative substitutions of the amino acid residues in a selected sequence, after aligning the amino acid sequences and introducing gaps in the candidate and/or selected sequences, if necessary, to achieve the maximum percent sequence similarity.

“Percent (%) identity” is defined herein as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps in the candidate and/or selected sequence, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative amino acid substitutions as part of the sequence identity.

The term “conservative substitution” as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic amino acid residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine, or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine and vice versa, of glutamic acid for aspartic acid, and vice versa, glutamine for asparagine, and vice versa, and the like. Neutral hydrophilic amino acids which can be substituted for one another include asparagine, glutamine, serine and threonine. The term “conservative substitution” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that the biologic activity of the peptide is maintained. Biological similarity between amino acid residues refers to similarities between properties such as, but not limited to, hydrophobicity, mutation frequency, charge, side chain length, size chain volume, pKa, polarity, aromaticity, solubility, surface area, peptide bond geometry, secondary structure propensity, average solvent accessibility, etc.

Alignment for purposes of determining percent homology (i.e., sequence similarity) or percent identity can be achieved in various ways that are within the skill in the art, for instance, using publicly or proprietary algorithms. For instance, sequence similarity can be determined using pairwise alignment methods, e.g., BLAST, BLAST-2, ALIGN, or ALIGN-2 or multiple sequence alignment methods such as Megalign (DNASTAR), ClustalW or T-Coffee software. Those skilled in the art can determine appropriate scoring functions, e.g., gap penalties or scoring matrices for measuring alignment, including any algorithms needed to achieve optimal alignment quality over the full-length of the sequences being compared. Furthermore, those skilled in the art would appreciate that methods to identify proteins with a certain fold, e.g., the FnIII fold, and to align the amino acid sequences of such proteins, include sequence-sequence methods, sequence-profile methods, and profile-profile methods. In addition, sequence alignment can be achieved using structural alignment methods (e.g., methods using secondary or tertiary structure information to align two or more sequences), or hybrid methods combining sequence, structural, and phylogenetic information to identify and optimally align candidate protein sequences.

A “protein sequence” or “amino acid sequence” means a linear representation of the amino acid constituents in a polypeptide in an amino-terminal to carboxyl-terminal direction in which residues that neighbor each other in the representation are contiguous in the primary structure of the polypeptide.

The term “nucleic acid” refers to any two or more covalently bonded nucleotides or nucleotide analogs or derivatives. As used herein, this term includes, without limitation, DNA, RNA, and PNA. “Nucleic acid” and “polynucleotide” are used interchangeably herein.

The term “polynucleotide” is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). The term “isolated” nucleic acid or polynucleotide is intended refers to a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding, e.g., a scaffold of the invention contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

The term “pharmaceutically acceptable” refers to a compound or protein that can be administered to an animal (for example, a mammal) without significant adverse medical consequences.

The term “physiologically acceptable carrier” refers to a carrier which does not have a significant detrimental impact on the treated host and which retains the therapeutic properties of the compound with which it is administered. One exemplary physiologically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences, (18^(th) edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa., incorporated herein by reference.

By a “polypeptide” is meant any sequence of two or more amino acids linearly linked by amide bonds (peptide bonds) regardless of length, post-translation modification, or function. “Polypeptide,” “peptide,” and “protein” are used interchangeably herein. Thus, peptides, dipeptides, tripeptides, or oligopeptides are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. A polypeptide can be generated in any manner, including by chemical synthesis.

Also included as polypeptides of the present invention are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. Variants can occur naturally or be non-naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions, or additions. Also included as “derivatives” are those peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.

The term “derived from [e.g., a protein or a polynucleotide]” means that a protein or polynucleotide is related to a reference protein or polynucleotide. The relation can be, for example, one of sequence or structural similarity. A protein or polynucleotide can be derived from a reference protein or polynucleotide via one or more of, e.g., mutation (e.g., deletion or substitution), chemical manipulation (e.g., chemical conjugation of a scaffold to PEG or to another protein), genetic fusion (e.g., genetic fusion of two or more scaffolds to a linker, a heterologous moiety, or combinations thereof), de novo synthesis based on sequence or structural similarity, or recombinant production in a heterologous organism.

By “randomized” or “mutated” is meant including one or more amino acid alterations, including deletion, substitution or addition, relative to a template sequence. By “randomizing” or “mutating” is meant the process of introducing, into a sequence, such an amino acid alteration. Randomization or mutation can be accomplished through intentional, blind, or spontaneous sequence variation, generally of a nucleic acid coding sequence, and can occur by any technique, for example, PCR, error-prone PCR, or chemical DNA synthesis. The terms “randomizing”, “randomized”, “mutating”, “mutated” and the like are used interchangeably herein.

By a “cognate” or “cognate, non-mutated protein” is meant a protein that is identical in sequence to a variant protein, except for the amino acid mutations introduced into the variant protein, wherein the variant protein is randomized or mutated.

By “RNA” is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. One example of a modified RNA included within this term is phosphorothioate RNA.

The terms “scaffold of the invention” or “scaffolds of the invention” as used herein, refers to multimeric scaffolds as well as monomeric FnIII scaffolds. The term “target” refers to a compound recognized by a specific scaffold of the invention. Typical targets include proteins, polysaccharides, polynucleotides and small molecules. The terms “target” and “antigen” are used interchangeably herein. The term “specificity” as used herein, e.g., in the terms “specifically binds” or “specific binding,” refers to the relative affinity by which a scaffold of the invention binds to one or more antigens via one or more antigen binding domains, and that binding entails some complementarity between one or more antigen binding domains and one or more antigens. According to this definition, a scaffold of the invention is said to “specifically bind” to an epitope when it binds to that epitope more readily than it would bind to a random, unrelated epitope.

The term “affinity” as used herein refers to a measure of the strength of the binding of a certain scaffold of the invention to an individual epitope.

The term “avidity” as used herein refers to the overall stability of the complex between a population of scaffolds of the invention and a certain epitope, i.e., the functionally combined strength of the binding of a plurality of scaffolds with the antigen. Avidity is related to both the affinity of individual antigen-binding domains with specific epitopes, and also the valency of the scaffold of the invention.

The term “action on the target” refers to the binding of a multimeric scaffold of the invention to one or more targets and to the biological effects resulting from such binding. In this respect, multiple antigen binding units in a multimeric scaffold can interact with a variety of targets and/or epitopes and, for example, bring two targets physically closer, trigger metabolic cascades through the interaction with distinct targets, etc.

The term “valency” as used herein refers to the number of potential antigen-binding modules, e.g., the number of FnIII modules in a scaffold of the invention. When a scaffold of the invention comprises more than one antigen-binding module, each binding module can specifically bind, e.g., the same epitope or a different epitope, in the same target or different targets.

The term “disulfide bond” as used herein includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.

The terms “Tn3 module” and “Tn3 scaffold” as used herein, refers to a FnIII scaffold wherein the A beta strand comprises SEQ ID NO: 42, the B beta strand comprises SEQ ID NO: 43, the C beta strand SEQ ID NO: 45 or 131, the D beta strand comprises SEQ ID NO: 46, the E beta strand comprises SEQ ID NO: 47, the F beta strand comprises SEQ ID NO: 49, and the beta strand G comprises SEQ ID NO: 52, wherein at least one loop is a non-naturally occurring variant of the loops in the “wild type Tn3 scaffold.” In certain embodiments, one or more of the beta strands of a Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand (e.g., the cysteine in SEQ ID NOs: 45 or 131) and F beta strands (SEQ ID NO: 49) are not substituted.

The term “wild type Tn3 scaffold” as used herein refers to an FnIII scaffold comprising SEQ ID NO: 1, i.e., an engineered FnIII scaffold derived from the 3^(rd) FnIII of human tenascin C.

The term “immunoglobulin” and “antibody” comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon. It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. Modified versions of each of these classes are readily discernable to the skilled artisan. As used herein, the term “antibody” includes but not limited to an intact antibody, a modified antibody, an antibody VL or VL domain, a CH1 domain, a Ckappa domain, a Clambda domain, an Fc domain (see supra), a CH2, or a CH3 domain.

As used herein, the term “modified antibody” includes synthetic forms of antibodies which are altered such that they are not naturally occurring, e.g., antibodies that comprise at least two heavy chain portions but not two complete heavy chains (as, e.g., domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) altered to bind to two or more antigens or to different epitopes of a single antigen). In addition, the term “modified antibody” includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that to three or more copies of the same antigen). (See, e.g., Antibody Engineering, Kontermann & Dubel, eds., 2010 Springer Protocols, Springer).

The terms “TRAIL R2” or “TRAIL R2 receptor” are used interchangeably herein to refer to the full length TRAIL receptor sequence and soluble, extracellular domain forms of the receptor described in Sheridan et al., Science, 277:818-821 (1997); Pan et al., Science, 277:815-818 (1997), U.S. Pat. Nos. 6,072,047 and 6,342,369: PCT Publ. Nos. WO98/51793, WO98/41629, WO98/35986, WO99/02653, WO99/09165, WO98/46643, and WO99/11791; Screaton et al., Curr. Biol., 7:693-696 (1997); Walczak et al., EMBO J., 16:5386-5387 (1997); Wu et al., Nature Genetics, 17:141-143 (1997). Representative full length TRAIL receptor sequences are available at GenBank Accession Nos. AAC51778.1 and 014763.2.

“TRAIL” or “TRAIL polypeptide” refers to a ligand that binds to one or more TRAIL receptors, including TRAIL R2, as well as biologically active fragments thereof. Representative TRAIL sequences are available at GenBank Accession Nos. AAH32722.1 and P50591.1.

The term “CD40L” refers to the CD40 ligand protein also known as CD154, gp39 or TBAM. CD40L a 33 kDa, Type II membrane glycoprotein. Additionally, shorter 18 kDa CD154 soluble forms exist, (also known as sCD40L). Representative human CD40L sequences are available at GenBank Accession No. AAA35662.1 and at UniProt Accession No. P29965. Representative murine CD40L sequences are available at GenBank Accession No. AAI19226.1 and at UniProt Accession No. P27548.

The term “in vivo half-life” is used in its normal meaning, i.e., the time at which 50% of the biological activity of a polypeptide is still present in the body/target organ, or the time at which the activity of the polypeptide is 50% of its initial value. As an alternative to determining functional in vivo half-life, “serum half-life” may be determined, i.e., the time at which 50% of the polypeptide molecules circulate in the plasma or bloodstream prior to being cleared. Determination of serum-half-life is often more simple than determining functional in vivo half-life and the magnitude of serum-half-life is usually a good indication of the magnitude of functional in vivo half-life. Alternative terms to serum half-life include plasma half-life, circulating half-life, circulatory half-life, serum clearance, plasma clearance, and clearance half-life. The functionality to be retained is normally selected from procoagulant, proteolytic, co-factor binding, receptor binding activity, or other type of biological activity associated with the particular protein.

The term “increased” with respect to the functional in vivo half-life or plasma half-life is used to indicate that the relevant half-life of the polypeptide is statistically significantly increased relative to that of a reference molecule (for example an unmodified polypeptide), as determined under comparable conditions. For instance the relevant half-life may be increased by at least about 25%, such as by at least about 50%, e.g., by at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 500% compared to an unmodified reference molecule. In other embodiments, the half-life may be increased by about at least 1 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, or at least 50 fold as compared to an unmodified reference molecule.

The term “expression” as used herein refers to a process by which a gene produces a biochemical, for example, a scaffold of the invention or a fragment thereof. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into one or more mRNAs, and the translation of such mRNAs into one or more polypeptides. If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors.

An “expression product” can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide. Expression products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

The term “vector” or “expression vector” is used herein to mean vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired expression product in a host cell. As known to those skilled in the art, such vectors can easily be selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired nucleic acid and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.

The term “host cells” refers to cells that harbor vectors constructed using recombinant DNA techniques and encoding at least one expression product. In descriptions of processes for the isolation of an expression product from recombinant hosts, the terms “cell” and “cell culture” are used interchangeably to denote the source of the expression product unless it is clearly specified otherwise, i.e., recovery of the expression product from the “cells” means either recovery from spun down whole cells, or recovery from the cell culture containing both the medium and the suspended cells.

The terms “treat” or “treatment” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder in a subject, such as the progression of an inflammatory disease or condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

The term “treatment” also means prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

The terms “subject,” “individual,” “animal,” “patient,” or “mammal” refer to any individual, patient or animal, in particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

Introduction

FnIII scaffolds derived from whole, stable, and soluble structural modules found in human body fluid proteins and from other sources in nature, including but not limited to, thermophilic bacteria and archaea, have been engineered to be superior both to antibody-derived fragments and to non-antibody frameworks. One particular example of scaffold engineering is the introduction of at least one non-naturally occurring intramolecular disulfide bond in an FnIII scaffold. In one embodiment, the multimeric scaffolds of the invention comprise tandem repeats of these FnIII scaffolds wherein at least one FnIII scaffold comprises one non-naturally occurring intramolecular disulfide bond. In some embodiments, the tandem scaffolds are fused by a peptide linker, thereby allowing expression as a single construct.

The FnIII scaffolds that make up the multimeric scaffolds correctly fold independently of each other, retain their binding specificity and affinity, and each of the scaffold domains retains its functional properties. When the FnIII scaffolds that make up the multimeric scaffolds are assembled in high valency multimeric scaffolds, e.g., hexavalent or octavalent scaffolds, the scaffolds correctly fold independently of each other, retain their binding specificity and affinity, and each of the scaffold domains retains its functional properties.

Multimeric scaffolds, including high valency scaffolds (e.g., hexavalent or octavalent), fold correctly even when the topology of construct is not linear, e.g., when the monomeric FnIII or multimeric FnIII scaffolds are assembled in complex branched structures (e.g., Fc fusion constructs or antibody-like constructs).

Native FnIII domains such as the 10th FnIII domain of human fibronectin (10FnIII) and the vast majority of naturally occurring FnIII domains contain no disulfide bonds or free cysteines. When multidomain proteins are engineered by introducing multiple cysteines, lack of protein expression, precipitation of the resulting proteins, or production of non-functional proteins, are common occurrences. These deleterious effects are due to the incorrect formation of intramolecular intradomain and/or interdomain disulfide bonds, and/or the incorrect formation of intermolecular disulfide bonds, which result in incorrect protein folding. These effects are generally intensified when the number of cysteines and/or protein domains is increased.

For example, a linear FnIII scaffold comprising 8 wild type Tn3 scaffolds (SEQ ID NO: 1) would contain 16 cysteines along a single polypeptide amino acid sequence. In another exemplary embodiment, an antibody-like construct comprising 4 Tn3 modules, wherein two Tn3 modules are linked to IgG heavy chains and two Tn3 are linked to IgG light chains, would comprise 32 cysteines distributed among 4 different polypeptide chains. Accordingly, it is highly unexpected that multimeric FnIII scaffolds comprising such number of cysteines and such structural complexity will fold correctly and display improved stability and target binding properties when compared to their respective FnIII monomeric domains.

When FnIII scaffolds comprising one or more engineered disulfide bridges are assembled in high valency multimeric formats, each individual monomer scaffold folds correctly retaining its binding specificity and affinity, as well as its functional properties. In addition, the monomeric scaffolds are capable of forming stable, functional, and correctly folded multimeric scaffolds.

An advantage of the multimeric scaffolds of the invention is their ability to bind to multiple epitopes, e.g., (i) binding to multiple epitopes in a single target, (ii) binding to a single epitope in multiple targets, (iii) binding to multiple epitopes located on different subunits of one target, or (iv) binding to multiple epitopes on multiple targets, thus increasing avidity.

In addition, due to the flexibility of the multimeric scaffolds and to the possibility of varying the distance between multiple FnIII modules via linkers, the multimeric scaffolds are capable of binding to multiple target molecules on a surface (either on the same cell/surface or in different cells/surfaces).

As a result of their ability to bind simultaneously to more than one target, the multimeric scaffolds of the invention can be used to modulate multiple pathways, cross-link receptors on a cell surface, bind cell surface receptors on separate cells, and/or bind target molecules or cells to a substrate.

From prior sequence analysis of FnIII domains, large variations were seen in the BC and FG loops, suggesting that these loops are not crucial to stability (see, for example, PCT Publication No: WO 2009/058379). The present invention provides FnIII scaffolds having improved stability, which vary in amino acid sequence but which comprise an FG loop having a shorter length than that of a FnIII domain/scaffold of interest. Although the amino acids sequences of FnIII domains tend to show low sequence similarity, their overall three dimensional structure is similar. Accordingly, using known techniques, such as sequence analysis and tertiary structure overlay, the specific locations of FG loops of FnIII scaffolds from different species and different proteins, even when overall sequence similarity is low, can be identified and be subjected to mutation. In some embodiments, the engineered FG loop has an amino acid sequence length that is at least one amino acid shorter than the length of the starting FG loop. It has been observed that shortening the FG loops results in a mutated FnIII scaffold that has increased stability. Consequently, another aspect of the invention provides FnIII variants having increased protein stability.

In certain embodiments, the scaffold of the invention comprises an FG loop having 9 amino acids and an increased stability compared to a scaffold comprising the native third FnIII domain of human tenascin C which has an FG loop length of 10 amino acids. Additionally the present invention provides libraries of diverse FnIII scaffolds having specified FG loop lengths which are useful for isolating FnIII scaffolds having increased stability as compared to a FnIII domain/scaffold of interest.

In addition, the present invention provides multispecific scaffolds that can bind to two or more different targets, affinity matured scaffolds wherein the affinity of a scaffold for a specific target is modulated via mutation, and scaffolds whose immunogenicity and/or cross-reactivity among animal species is modulated via mutation. Also, the invention provides methods to produce the scaffolds of the invention as well as methods to engineer scaffolds with desirable physicochemical, pharmacological, or immunological properties. Furthermore, the present invention provides uses for such scaffolds and methods for therapeutic, prophylactic, and diagnostic use.

The FnIII Structural Motif

The scaffolds of the present invention are based on the structure of a fibronectin module of type III (FnIII), a domain found widely across all three domains of life and viruses, and in multitude of protein classes. The FnIII domain is found in fibronectins, multidomain-proteins found in soluble form in blood plasma and in insoluble form in loose connective tissue and basement proteins This domain is found in numerous proteins sequenced to date. The FnIII domain superfamily represents at least 45 different protein families, the majority of which are involved in cell surface binding in some manner, or function as receptors. Specific examples of proteins containing FnIII domains include fibronectins, tenascins, intracellular cytoskeletal proteins, cytokine receptors, receptor protein tyrosine kinases, and prokaryotic enzymes (Bork and Doolittle, Proc. Natl. Acad. Sci. USA 89:8990-8894, 1992; Bork et al., Nature Biotechnol. 15:553-557, 1997; Meinke et al., J. Bacteriol. 175:1910-1918, 1993; Watanabe et al., J. Biol. Chem. 265:15659-15665, 1990).

Naturally occurring protein sequences comprising FnIII domains include but are not limited to fibronectin, tenascin C, growth hormone receptor, β-common receptor, IL-5R, tenascin XB, and collagen type XIV. Although the domain appears widely distributed in nature, the percentage of amino acid sequence similarity between the amino acid sequences of highly divergent FnIII domains can be very low.

In specific embodiments, the scaffolds of the invention are derived from the third FnIII domain of human tenascin C (SEQ ID NO: 4). In one specific embodiment, the scaffolds of the invention comprise a Tn3 module. The overall three dimensional fold of this domain is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain (VH), which in the single domain antibodies of camels and camelids (e.g., llamas) comprises the entire antigen recognition unit.

The FnIII scaffolds of the invention and the native FnIII domains are characterized by the same three dimensional structure, namely a beta-sandwich structure with three beta strands (A, B, and E) on one side and four beta strands (C, D, F, and G) on the other side, connected by six loop regions. These loop regions are designated according to the beta-strands connected to the N- and C-terminus of each loop. Accordingly, the AB loop is located between beta strands A and B, the BC loop is located between strands B and C, the CD loop is located between beta strands C and D, the DE loop is located between beta strands D and E, the EF loop is located between beta strands E and F, and the FG loop is located between beta strands F and G. FnIII domains possess solvent exposed loops tolerant of randomization, which facilitates the generation of diverse pools of protein scaffolds capable of binding specific targets with high affinity.

The multiple sequence alignment shown in FIG. 16 identifies the positions of the beta strands and loops for numerous native FnIII domains based on the analysis of their three dimensional structures and amino acid sequences. These FnIII domains can be utilized to design proteins which are capable of binding to virtually any target compound, for example, any protein of interest. One skilled in the art will appreciate that the alignment shown in FIG. 16 is exemplary and non-limiting. For example, the alignment of FIG. 16 may be expanded by incorporating protein domains recognized to contain the Interpro IPRO08957 fibronectin type III domain signature as determined using the InterProScan program, or recognized to contain the Pfam PF00041 fibronectin type III domain signature as determined using Pfam_scan, HMMER, or any other program capable of comparing a protein sequence to a Hidden Markov model.

Thus protein scaffold engineering and design can be based on, e.g.,

(i) the aligned sequence set shown in FIG. 16,

(ii) a subset of aligned sequences derived from FIG. 16,

(iii) a different aligned set comprising amino acid sequences of FnIII domains

-   -   whose three dimensional structure has been determined         experimentally (e.g., through the use of X-ray diffraction         crystallography or NMR), and/or     -   (iv) amino acid sequences of FnIII domains whose three         dimensional structure is not yet available but recognized to         contain the Interpro IPRO08957 fibronectin type III domain         signature as determined using the InterProScan program, or         recognized to contain the Pfam PF00041 fibronectin type III         domain signature as determined using Pfam_scan, HMMER, or any         other program capable of comparing a protein sequence to a         Hidden Markov model.

In one aspect of the invention, FnIII domains are used as scaffolds which are subjected to directed evolution designed to randomize one or more of the loops which are analogous to the complementarity-determining regions (CDRs) of an antibody variable region. Such a directed evolution approach results in the production of antibody-like molecules with high affinities for targets of interest. In addition, in some embodiments the scaffolds described herein can be used to display defined exposed loops (for example, loops previously randomized and selected on the basis of target binding) in order to direct the evolution of molecules that bind to such introduced loops. This type of selection can be carried out to identify recognition molecules for any individual CDR-like loop or, alternatively, for the recognition of two or all three CDR-like loops combined into a nonlinear epitope binding moiety.

In some embodiments, the scaffolds of the invention are molecules based on the third FnIII domain of human tenascin C structural motif described in PCT Publication No: WO 2009/058379. A set of three loops (designated BC, DE, and FG), which can confer specific target binding, run between the B and C strands; the D and E strands, and the F and G beta strands, respectively. The BC, DE, and FG loops of the third FnIII domain of human tenascin C are 9, 6, and 10 amino acid residues long, respectively. The length of these loops falls within the narrow range of the cognate antigen-recognition loops found in antibody heavy chains, that is, 7-10, 4-8, and 4-28 amino acids in length, respectively. Similarly, a second set of loops, the AB, CD, and EF loops (7, 7, and 8, amino acids in length respectively) run between the A and B beta strands; the C and D beta strands; and the E and F beta strands, respectively.

In other embodiments, molecules based on the tenth FnIII (“10FnIII”) domain derived from human fibronectin (SEQ ID NO: 54) can be used as scaffolds. As defined in FIG. 16, in the tenth FnIII domain of human fibronectin the AB loop corresponds to SEQ ID NO: 55, the BC loop corresponds to SEQ ID NO:56, the CD loop corresponds to SEQ ID NO: 57, the DE loop corresponds to SEQ ID NO: 58, the EF loop corresponds to SEQ ID NO: 59, and the FG loop corresponds to SEQ ID NO: 60. It will be understood that alternative definitions for these regions are known in the art, see for example, Xu et al. Chemistry & Biology 9:933-942, 2002, which may be used as described herein.

In still other embodiments, molecules based on the fourteenth FnIII (“14FnIII”) domain derived from human fibronectin (SEQ ID NO: 69) can be used as scaffolds. As defined in FIG. 16, the AB loop of 14FnIII corresponds to SEQ ID NO: 70, the BC loop corresponds to SEQ ID NO: 71, the CD loop corresponds to SEQ ID NO: 72, the DE loop corresponds to SEQ ID NO: 73, the EF loop corresponds to SEQ ID NO: 74, and the FG loop corresponds to SEQ ID NO: 75. It will be understood that alternative definitions for these regions are known in the art, see for example, Cappuccilli et al. (U.S. Patent Publication No. 2009/0176654) which may be used as described herein.

In still other embodiments, molecules based on a consensus sequence derived from the sequence of FnIII domains of Tenascin (SEQ ID NO: 256) can be used as scaffolds. The loops of a Tenascin consensus FnIII are defined in Table 1, the AB loop corresponds to SEQ ID NO: 257, the BC loop corresponds to SEQ ID NO: 258, the CD loop corresponds to SEQ ID NO: 259, the DE loop corresponds to SEQ ID NO: 260, the EF loop corresponds to SEQ ID NO: 261, and the FG loop corresponds to SEQ ID NO: 262. It will be understood that alternative definitions for these regions are known in the art, see for example, Jacobs et al. (International Patent Publication No. WO 2010/093627) which may be used as described herein.

Once randomized and selected for high affinity binding to a target, the loops in the FnIII domain may make contacts with targets equivalent to the contacts of the cognate CDR loops in antibodies. Accordingly, in some embodiments the AB, CD, and EF loops, alone or in combination, are randomized and selected for high affinity binding to one or more targets. In some embodiments, this randomization and selection process may be performed in parallel with the randomization of one or more of the BC, DE, and FG loops, whereas in other embodiments this randomization and selection process is performed in series.

Monomeric Scaffolds of the Invention

The invention provides recombinant, non-naturally occurring FnIII scaffolds comprising, a plurality of beta strand domains linked to a plurality of loop regions, wherein one or more of said loop regions vary by deletion, substitution or addition of at least one amino acid from the cognate loops in a FnIII domain/scaffold of interest (referred to herein as an “FOI”), and wherein the beta strands of the FnIII scaffold have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more homology (i.e., sequence similarity) to the cognate beta strands of the FOI.

The FOI is a reference used for comparing sequence, physicochemical and/or phylogenetic characteristics. It will be understood that, when comparing the sequence of a scaffold of the invention to the sequence of an FOI, the same definition of the beta strands and loops is utilized. The FOI can be a native FnIII domain, a scaffold comprising a native FnIII domain or a non-naturally occurring FnIII scaffold. In certain embodiments, the FOI comprises at least one non-naturally occurring loop. In certain embodiments, the FOI comprises at least one non-naturally occurring beta strand. In certain embodiments, the FOI comprises at least one non-naturally occurring loop and at least one non-naturally occurring beta strand. In certain embodiments, the FOI comprises at least one non-naturally occurring disulfide bond. In a specific embodiment, the FOI comprises a wild type Tn3 scaffold (SEQ ID NO:1), a scaffold derived from the third FnIII domain of human tenascin that contains an engineered intramolecular disulfide bond.

In a specific embodiment, the monomer scaffolds of the invention comprise seven beta strands, designated A, B, C, D, E, F, G, linked to six loop regions, designated AB, BC, CD, DE, EF, FG, wherein at least one loop is a non-naturally occurring variant of the cognate loop in an FOI and the beta strands have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more homology (i.e., sequence similarity) to the cognate beta strands of the FOI.

In a specific embodiment, the monomer scaffolds of the invention comprise seven beta strands, designated A, B, C, D, E, F, G, linked to six loop regions, designated AB, BC, CD, DE, EF, FG, wherein at least one loop is a non-naturally occurring variant of the cognate loop in an FOI and the beta strands have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identity to the cognate domain of the FOI.

In a specific embodiment, the FOI comprises a third FnIII domain of human tenascin C (SEQ ID NO: 4). In one embodiment, the scaffolds of the invention comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more homology (i.e., sequence similarity) to the third FnIII domain of human tenascin C (SEQ ID NO:4).

In one embodiment, the scaffolds of the invention comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identity to the third FnIII domain of human tenascin C (SEQ ID NO:4).

In another embodiment, the monomer scaffolds of the invention comprise the amino acid sequence:

IEV(X_(AB))_(n)ALITW(X_(BC))_(n)CELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n)YEVSLIC(X_(FG))_(n)KET FTT, wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG) represent the amino acid residues present in the AB, BC, CD, DE, EF, and FG loops, respectively, wherein X₁ represents amino acid residue A or T, and wherein n=2-26.

In another embodiment, the monomer scaffolds of the invention comprise the amino acid sequence:

IEV(X_(AB))_(n)ALITW(X_(BC))_(n)IELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n)YEVSLIS(X_(FG))_(n)KETF TT, wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG) represent the amino acid residues present in the AB, BC, CD, DE, EF, and FG loops, respectively, wherein X₁ represents amino acid residue A or T, and wherein n=2-26.

In one embodiment, X_(AB) consists of SEQ ID NO: 35. In one embodiment, X_(BC) consists of SEQ ID NO: 36. In one embodiment, X_(CD) consists of SEQ ID NO: 37. In one embodiment, X_(DE) consists of SEQ ID NO: 38. In one embodiment, X_(EF) consists of SEQ ID NO: 39. In one embodiment, X_(FG) consists of SEQ ID NO: 40.

In one embodiment, X_(AB) comprises SEQ ID NO: 35. In one embodiment, X_(BC) comprises SEQ ID NO: 36. In one embodiment, X_(CD) comprises SEQ ID NO: 37. In one embodiment, X_(DE) comprises SEQ ID NO: 38. In one embodiment, X_(EF) comprises SEQ ID NO: 39. In one embodiment, X_(FG) comprises SEQ ID NO: 40.

In certain embodiments, X_(AB) consists of SEQ ID NO: 35, X_(CD) consists of SEQ ID NO: 37, and X_(EF) consists of SEQ ID NO: 39. In one embodiment, X_(BC) consists of SEQ ID NO: 36, X_(DE) consists of SEQ ID NO: 38, and X_(FG) consists of SEQ ID NO: 40.

In certain embodiments, X_(AB) comprises SEQ ID NO: 35, X_(CD) comprises SEQ ID NO: 37, and X_(EF) comprises SEQ ID NO: 39. In one embodiment, X_(BC) comprises SEQ ID NO: 36, X_(DE) comprises SEQ ID NO: 38, and X_(FG) comprises SEQ ID NO: 40.

In a specific embodiment, the FOI comprises a wild type tenth fibronectin type III domain (10FnIII) of human fibronectin scaffold (SEQ ID NO: 54). In one embodiment, the scaffolds of the invention comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more similarity to wild type 10FnIII (SEQ ID NO: 54).

In one embodiment, the monomer scaffolds of the invention comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identity to wild type 10FnIII (SEQ ID NO: 54).

In another embodiment, the monomer scaffolds of the invention comprise the amino acid sequence:

LEV(X_(AB))_(n)LLISW(X_(BC))_(n)YRITYGE(X_(CD))_(n)QEFTV(X_(DE))_(n)ATI(X_(EF))_(n)YTITVYA (X_(FG))_(n)SINYRT, wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG) represent the amino acid residues present in the AB, BC, CD, DE, EF, and FG loops, and wherein n=2-26.

In one embodiment, X_(AB) is the amino acid sequence of loop AB of 10FnIII (SEQ ID NO: 55). In one embodiment, X_(BC) is the amino acid sequence of loop BC of wild type 10FnIII (SEQ ID NO: 56). In one embodiment, X_(CD) is the amino acid sequence of loop CD of wild type 10FnIII (SEQ ID NO: 57). In one embodiment, X_(DE) is the amino acid sequence of loop DE of wild type 10FnIII (SEQ ID NO: 58). In one embodiment, X_(EF) is the amino acid sequence of loop EF of wild type 10FnIII (SEQ ID NO: 59). In one embodiment, X_(FG) is the amino acid sequence of loop FG of wild type 10FnIII (SEQ ID NO: 60).

In certain embodiments, X_(AB) is the amino acid sequence of loop AB of wild type 10FnIII (SEQ ID NO: 55), X_(C)D is the amino acid sequence of loop CD of wild type 10FnIII (SEQ ID NO: 57), and X_(EF) is the amino acid sequence of loop EF of wild type 10FnIII (SEQ ID NO: 59).

In one embodiment, X_(BC) is the amino acid sequence of loop BC of wild type 10FnIII (SEQ ID NO: 56), X_(DE) is the amino acid sequence of loop DE of wild type 10FnIII (SEQ ID NO: 58), and X_(FG) is the amino acid sequence of loop FG of wild type 10FnIII (SEQ ID NO: 60).

In a specific embodiment, the FOI comprises a wild type fourteenth type III fibronectin domain (14FnIII) of human fibronectin scaffold (SEQ ID NO: 69). In one embodiment, the scaffolds of the invention comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more similarity to wild type 14FnIII (SEQ ID NO: 69).

In one embodiment, the monomer scaffolds of the invention comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identity to wild type 14FnIII (SEQ ID NO: 69).

In another embodiment, the monomer scaffolds of the invention comprise the amino acid sequence:

ARV(X_(AB))_(n)ITISW(X_(BC))_(n)FQVDAVP(X_(CD))_(n)IQRTI(X_(DE))_(n)YTI(X_(EF))_(n)YKIYLYT (X_(FG))_(n)VIDAST, wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG) represent the amino acid residues present in the AB, BC, CD, DE, EF, and FG loops, respectively, and wherein n=2-26.

In one embodiment, X_(AB) is the amino acid sequence of loop AB of wild type 14FnIII (SEQ ID NO: 70). In one embodiment, X_(BC) is the amino acid sequence of loop BC of wild type 14FnIII (SEQ ID NO: 71). In one embodiment, X_(CD) is the amino acid sequence of loop CD of wild type 14FnIII (SEQ ID NO: 72). In one embodiment, X_(DE) is the amino acid sequence of loop DE of wild type 14FnIII (SEQ ID NO: 73). In one embodiment, X_(EF) is the amino acid sequence of loop EF of wild type 14FnIII (SEQ ID NO: 74). In one embodiment, X_(FG) is the amino acid sequence of loop FG of wild type 14FnIII (SEQ ID NO: 75).

In certain embodiments, X_(A)B is the amino acid sequence of loop AB of wild type 14FnIII (SEQ ID NO: 70), X_(CD) is the amino acid sequence of loop CD of wild type 14FnIII (SEQ ID NO: 72), and X_(EF) is the amino acid sequence of loop EF of wild type 14FnIII (SEQ ID NO: 74). In one embodiment, X_(BC) is the amino acid sequence of loop BC of wild type 14FnIII (SEQ ID NO: 71), X_(DE) is the amino acid sequence of loop DE of wild type 14FnIII (SEQ ID NO: 73), and X_(FG) is the amino acid sequence of loop FG of wild type 14FnIII (SEQ ID NO: 75).

In a specific embodiment, the FOI comprises Tenascin consensus FnIII (SEQ ID NO: 256). In one embodiment, the scaffolds of the invention comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more similarity to Tenascin consensus FnIII (SEQ ID NO: 256).

In one embodiment, the monomer scaffolds of the invention comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identity to Tenascin consensus FnIII (SEQ ID NO: 256).

In another embodiment, the monomer scaffolds of the invention comprise the amino acid sequence:

LVV(X_(AB))_(n)LRLSW(X_(BC))_(n)FLIQYQE(X_(CD))_(n)INLTV(X_(DE))_(n)YDL(X_(EF))_(n)YTVSIYG(X_(FG))_(n)SA EFTT, wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG) represent the amino acid residues present in the AB, BC, CD, DE, EF, and FG loops, respectively, and wherein n=2-26.

In one embodiment, X_(AB) is the amino acid sequence of AB loop of Tenascin consensus FnIII (SEQ ID NO: 257). In one embodiment, X_(BC) is the amino acid sequence of loop BC of Tenascin consensus FnIII (SEQ ID NO: 258). In one embodiment, X_(CD) is the amino acid sequence of loop CD of Tenascin consensus FnIII (SEQ ID NO: 259). In one embodiment, X_(DE) is the amino acid sequence of loop DE of Tenascin consensus FnIII (SEQ ID NO: 260). In one embodiment, X_(EF) is the amino acid sequence of loop EF of Tenascin consensus FnIII (SEQ ID NO: 261). In one embodiment, X_(FG) is the amino acid sequence of loop FG of Tenascin consensus FnIII (SEQ ID NO: 262).

In certain embodiments, X_(AB) is the amino acid sequence of loop AB of Tenascin consensus FnIII (SEQ ID NO: 257), X_(CD) is the amino acid sequence of loop CD of Tenascin consensus FnIII (SEQ ID NO: 259), and X_(EF) is the amino acid sequence of loop EF of Tenascin consensus FnIII (SEQ ID NO: 261). In one embodiment, X_(BC) is the amino acid sequence of loop BC of Tenascin consensus FnIII (SEQ ID NO: 258), X_(DE) is the amino acid sequence of loop DE of Tenascin consensus FnIII (SEQ ID NO: 260), and X_(FG) is the amino acid sequence of loop FG of Tenascin consensus FnIII (SEQ ID NO: 262).

In another embodiment, the monomer scaffolds of the invention comprise the amino acid sequence selected from the group consisting of:

IEV(X_(AB))_(n)ALITW(X_(BC))_(n)IELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n)YEVSLIS(X_(FG))_(n)KETF TT, LEV(X_(AB))_(n)LLISW(X_(BC))_(n)YRITYGE(X_(CD))_(n)QEFTV(X_(DE))_(n)ATI(X_(EF))_(n)YTITVYA (X_(FG))_(n)SINYRT, ARV (X_(AB))_(n)ITISW(X_(BC))_(n)FQVDAVP(X_(CD))_(n)IQRTI(X_(DE))_(n)YTI(X_(EF))_(n)YKIYLYT(X_(FG))_(n)VID AST, and LVV(X_(AB))_(n)LRLSW(X_(BC))_(n)FLIQYQE(X_(CD))_(n)INLTV(X_(DE))_(n)YDL(X_(EF))_(n)YTVSIYG(X_(FG))_(n)SA EFTT, wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG) represent the amino acid residues present in the AB, BC, CD, DE, EF, and FG loops, respectively, wherein X₁ represents amino acid residue A or T, wherein n=2-26, and wherein: X_(AB) is selected from the group consisting of SEQ ID NOs: 35, 55, 70, or 257, X_(CD) is selected from the group consisting of SEQ ID NOs: 37, 57, 72, or 259, and X_(EF) is selected from the group consisting of SEQ ID NOs: 39, 59, 74, or 261.

In another embodiment, the monomer scaffolds of the invention comprise the amino acid sequence selected from the group consisting of:

IEV(X_(AB))_(n)ALITW(X_(BC))_(n)IELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n)YEVSLIS(X_(FG))_(n)KETF TT, LEV(X_(AB))_(n)LLISW(X_(BC))_(n)YRITYGE(X_(CD))_(n)QEFTV(X_(DE))_(n)ATI(X_(EF))_(n)YTITVYA (X_(FG))_(n)SINYRT, ARV(X_(AB))_(n)ITISW(X_(BC))_(n)FQVDAVP(X_(CD))_(n)IQRTI(X_(DE))_(n)YTI(X_(EF))_(n)YKIYLYT(X_(FG))_(n)VID AST, and LVV(X_(AB))_(n)LRLSW(X_(BC))_(n)FLIQYQE(X_(CD))_(n)INLTV(X_(DE))_(n)YDL(X_(EF))_(n)YTVSIYG(X_(FG))_(n)SA EFTT, wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG) represent the amino acid residue present in the AB, BC, CD, DE, EF, and FG loops, respectively, wherein X₁ represent amino acid residue A or T, wherein n=2-26, and wherein: X_(BC) is selected from the group consisting of SEQ ID NOs: 36, 56, 71, or 258, X_(DE) is selected from the group consisting of SEQ ID NOs: 38, 58, 73, or 260, and X_(FG) is selected from the group consisting of SEQ ID NOs: 40, 60, 75, or 262.

In other embodiments, the scaffolds of the invention comprise a Tn3 module. In still other embodiments, scaffolds of the invention comprise a Tn3 module (SEQ ID NO: 1), wherein beta strand C of a third FnIII domain of human tenascin C (SEQ ID NO; 44) is replaced by a variant beta strand C (SEQ ID NO: 45, or 131) comprising an N-terminal cysteine and wherein beta strand F of a third FnIII domain of human tenascin C (SEQ ID NO: 48) is replaced by a variant beta strand F (SEQ ID NO: 49) comprising a C-terminal cysteine. In some embodiments the scaffolds of the invention comprise a Tn3 module wherein one or more of the beta strands comprise at least one amino acid substitution except that the cysteine residues in the C and F beta strands (SEQ ID NOs: 45, or 131 and 49, respectively) may not be substituted. In certain embodiments, the scaffolds of the invention comprise a variant of a 10FnIII module, wherein one or more of the beta strands comprise at least one amino acid substitution. In other embodiments, the scaffolds of the invention comprise a variant of a 14FnIII module, wherein one or more of the beta strands comprise at least one amino acid substitution. In still other embodiments, the scaffolds of the invention comprise a variant of Tenascin consensus FnIII module, wherein one or more of the beta strands comprise at least one amino acid substitution.

In other embodiments, the naturally occurring sequence is a protein sequence corresponding to an additional FnIII domain from human tenascin C. In other embodiments, the naturally occurring sequence is a protein sequence corresponding to a FnIII domain from another tenascin protein including but not limited to the 29th FnIII domain from human tenascin XB (SEQ ID NO: 11), the 31st FnIII domain from human tenascin XB (SEQ ID NO:12), or the 32nd FnIII domain from human tenascin XB (SEQ ID NO: 13). In other embodiments, the naturally occurring sequence is a protein sequence corresponding to an FnIII domain from another organism (such as, but not limited to, murine, porcine, bovine, or equine tenascins).

In additional embodiments, FnIII domains used to generate scaffolds of the invention, include, e.g., related FnIII domains from animals, plants, bacteria, archaea, or viruses. Different FnIII domains from different organisms and parent proteins can be most appropriate for different applications; for example, in designing a scaffold stable at a low pH, it can be most desirable to generate that protein from organism that optimally grows at a low pH (such as, but not limited to Sulfolobus tokodaii). In another embodiment, related FnIII domains can be identified and utilized from thermophilic and hyperthermophilic organisms (e.g., hyperthermophilic bacteria or hyperthermophilic archaea). In some embodiments, FnIII domains used to generate scaffolds of the invention are FnIII domains from hyperthermophilic archaea such as, but not limited to, Archaeoglobus fulgidus and Staphylothermus marinus, each of which exhibit optimal growth at greater than 70° C. In other embodiments, the naturally occurring sequence corresponds to a predicted FnIII domain from a thermophilic organism, for example, but not limited to Archaeoglobus fulgidus, Staphylothermus marinus, Sulfolobus acidocaldarius, Sulfolobus solfataricus, and Sulfolobus tokodaii. In yet another embodiment, the scaffolds of the invention comprise a protein sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homology (sequence similarity) to any of the sequences from a sequence corresponding to a FnIII domain or a predicted FnIII domain from a thermophilic organism as described above. In some embodiments, the FnIII domains from thermophilic organisms are selected from the amino acid sequences of SEQ ID NOs: 20-33.

The loops connecting the various beta strands of the scaffolds of the invention can be randomized for length and/or sequence diversity. In one embodiment, the scaffolds of the invention have at least one loop that is randomized for length and/or sequence diversity. In one embodiment, at least one, at least two, at least three, at least four, at least five or at least six loops of a scaffold are randomized for length and/or sequence diversity. In one embodiment, at least one loop of the scaffolds of the invention is kept constant while at least one additional loop is randomized for length and/or sequence diversity. In another embodiment, at least one, at least two, or all three of loops AB, CD, and EF are kept constant while at least one, at least two, or all three of loops BC, DE, and FG are randomized for length or sequence diversity. In another embodiment, at least one, at least two, or at least all three of loops AB, CD, and EF are randomized while at least one, at least two, or all three of loops BC, DE, and FG are randomized for length and/or sequence diversity. In still another embodiment, at least one, at least two, at least three of loops, at least 4, at least five, or all six of loops AB, CD, EF, BC, DE, and FG are randomized for length or sequence diversity.

In some embodiments, one or more residues within a loop are held constant while other residues are randomized for length and/or sequence diversity. In some embodiments, one or more residues within a loop are held to a predetermined and limited number of different amino acids while other residues are randomized for length and/or sequence diversity. Accordingly, scaffolds of the invention can comprise one or more loops having a degenerate consensus sequence and/or one or more invariant amino acid residues. In one embodiment, the scaffolds of the invention comprise an AB loop which is randomized with the following consensus sequence: K-X-X-X-X-X-a, wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, and wherein (a) represents serine, threonine, alanine, or glycine. In another embodiment, the scaffolds of the invention comprise an AB loop which is randomized with the following consensus sequence: K-X-X-X-X-X-X-X-a, wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, and wherein (a) represents serine, threonine, alanine, or glycine.

In another embodiment, the scaffolds of the invention comprise a BC loop which is randomized with the following consensus sequence: S-X-a-X-b-X-X-X-G, wherein X represents any amino acid, wherein (a) represents proline or alanine and wherein (b) represents alanine or glycine. In another embodiment, the scaffolds of the invention comprise a BC loop which is randomized with the following consensus sequence: S-P-c-X-X-X-X-X-X-T-G, wherein X represents any amino acid and wherein (c) represents proline, serine or glycine. In still another embodiment, the scaffolds of the invention comprise a BC loop which is randomized with the following consensus sequence: A-d-P-X-X-X-e-f-X-I-X-G, wherein X represents any amino acid, wherein (d) represents proline, glutamate or lysine, wherein (e) represents asparagine or glycine, and wherein (f) represents serine or glycine.

In one embodiment, the scaffolds of the invention comprise a CD loop which is randomized with the following consensus sequence: X_(n), wherein X represents any amino acid, and wherein n=6, 7, 8, 9, or 10. In another embodiment, the scaffolds of the invention comprise an CD loop which is randomized with the following consensus sequence: X_(n), wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, and wherein n=7, 8, or 9.

In one embodiment, the scaffolds of the invention comprise an DE loop which is randomized with the following consensus sequence: X-X-X-X-X-X, wherein X represents any amino acid.

In one embodiment, the scaffolds of the invention comprise an EF loop which is randomized with the following consensus sequence: X-b-L-X-P-X-c-X, wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, wherein (b) represents asparagine, lysine, arginine, aspartic acid, glutamic acid, or glycine, and wherein (c) represents isoleucine, threonine, serine, valine, alanine, or glycine

In one embodiment, the scaffolds of the invention comprise an FG loop which is randomized with the following consensus sequence: X-a-X-X-G-X-X-X-b, wherein X represents any amino acid, wherein (a) represents asparagine, threonine or lysine, and wherein (b) represents serine or alanine. In another embodiment, the scaffolds of the invention comprise an FG loop which is randomized with the following consensus sequence: X-X-X-X-X-X-X-X-X (X₉), wherein X represents any amino acid. In still another embodiment, the scaffolds of the invention comprise an FG loop which is randomized with the following consensus sequence: X-a-X-X-X-X-b-N-P-A, wherein X represents any amino acid, wherein (a) represents asparagine, threonine or lysine and wherein (b) represents serine or glycine. In a specific embodiment, the scaffolds of the invention comprise an FG loop which is randomized with the following consensus sequence: X-a-X-X-G-X-X-S-N-P-A, wherein X represents any amino acid, and wherein (a) represents asparagine, threonine or lysine.

In certain embodiments, the scaffolds of the invention comprise an FG loop which is held to be at least one amino acid residue shorter than the cognate FG loop of an FOI and is further randomized at one or more positions. For example, as defined in FIG. 16 the native FG loop of the third FnIII domain of human tenascin C comprises 10 amino acid residues, accordingly, the FG loop would be held to 9 amino acid residues or less.

In some embodiments, a scaffold of the invention is a chimeric scaffold comprising one or more beta strands comprising amino acid sequences selected from homologous beta strands selected from a plurality of FOIs. In some embodiments, a scaffold of the invention is a chimeric scaffold wherein at least one of the loops BC, DE, and FG are randomized. In some embodiments, a scaffold of the invention is a chimeric scaffold wherein at least one of loops AB, CD, and EF is randomized.

In specific embodiments, at least one of loops BC, DE, and FG is randomized, wherein the A beta strand comprises SEQ ID NO:41, 42, 61, 62, 76, 77, 248 or 249, the B beta strand comprises SEQ ID NO:43, 63, 78, or 250, the C beta strand comprises SEQ ID NO:44, 45, 64, 79, 131, or 251, the D beta strand comprises SEQ ID NO:46, 65, 80, or 252, the E beta strand comprises SEQ ID NO:47, 66, 81, or 253, the F beta strand comprises SEQ ID NO:48, 49, 50, 51, 67, 82, or 254, and the G beta strand comprises SEQ ID NO:52, 53, 68, 83, or 255, the AB loop comprises SEQ ID NO:35, 55, 70, or 242, the CD loop comprises SEQ ID NO:37, 57, 72, or 244, and the EF loop comprises SEQ ID NO:39, 59, 74, or 246.

In other specific embodiments, at least one of loops AB, CD, and EF are randomized, wherein the A beta strand comprises SEQ ID NO:41, 42, 61, 62, 76, 77, 248 or 249, the B beta strand comprises SEQ ID NO:43, 63, 78, or 250, the C beta strand comprises SEQ ID NO:44, 45, 64, 79, 131, or 251, the D beta strand comprises SEQ ID NO:46, 65, 80, or 252, the E beta strand comprises SEQ ID NO:47, 66, 81 or 253, the F beta strand comprises SEQ ID NO:48, 49, 50, 51, 67, 82, or 254, and the G beta strand comprises SEQ ID NO:52, 53, 68, 83, or 255, the BC loop comprises SEQ ID NO:36, 56, 71, or 243, the DE loop comprises SEQ ID NO:38, 58, 73, 245 and the FG loop comprises SEQ ID NO:40, 60, 75, or 247.

Enhanced Scaffold Stability Non-Naturally Occurring Disulfide Bonds

The stability of scaffolds of the invention may be increased by many different approaches. In some embodiments, scaffolds of the invention can be stabilized by elongating the N- and/or C-terminal regions. The N- and/or C-terminal regions can be elongated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 amino acids. In other embodiments, the scaffolds of the invention can be stabilized by introducing an alteration that increases serum half-life, as described herein. In yet another embodiment, the scaffolds of the invention comprise an addition, deletion or substitution of at least one amino acid residue to stabilize the hydrophobic core of the scaffold.

Scaffolds of the invention also can be effectively stabilized by engineering non-natural disulfide bonds. Such engineered scaffolds can be efficiently expressed as part of multimeric scaffolds. The correct formation of the disulfide bonds and the correct folding of the engineered scaffold are evidenced by the preservation of the biological activity of the scaffold. The fact that an engineered scaffold comprising non-natural disulfide bonds can bind simultaneously to at least two targets (see, e.g., Example 8) or three targets (see, e.g., Example 12) provides evidence that the three dimensional structure of the scaffold is not significantly altered by the engineered disulfide bonds and that the relative positions of the target-binding loops are preserved. In some embodiments, scaffolds of the invention comprise non-naturally occurring disulfide bonds, as described in PCT Publication No: WO 2009/058379. A bioinformatics approach may be utilized to identify candidate positions suitable for engineering disulfide bonds.

In one embodiment, a monomeric scaffold of the invention comprise at least one, at least two, at least three, at least four, or at least five non-naturally occurring intramolecular disulfide bonds. In a specific embodiment, the invention provides a method of obtaining a scaffold having increased stability as compared to an FOI comprising two, three, four, or more engineered intramolecular disulfide bonds.

In one embodiment, the scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein said at least one non-naturally occurring disulfide bond stabilizes a monomer scaffold. In another embodiment, the scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond located between two beta strands within the same monomer scaffold. For example, within a monomer scaffold, at least one non-naturally occurring intramolecular disulfide bond can form a link between the A strand and B strand, or between the D strand and E strand, or between the F strand and G strand, or between the C strand and F strand.

In another embodiment, non-naturally occurring disulfide bonds form a first bond between the F strand and the G strand, and a second link between the C strand and F strand within a single monomer scaffold. In another embodiment, the scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond located between two loops in the same monomer scaffold. In another embodiment, the scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond located between a loop and a beta strand of the same monomer scaffold. In another embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond that is located within the same beta strand in a monomer scaffold. In another embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond that is located within the same loop in a monomer scaffold.

In another embodiment, scaffolds of the invention comprise at least one non-naturally occurring disulfide bond, wherein the bond is located between two distinct monomer scaffolds in a multimeric scaffold. In yet another embodiment, scaffolds of the invention comprise at least one non-naturally occurring disulfide bond, wherein the bond is located between two distinct multimeric scaffolds, i.e., the disulfide bond is an intermolecular disulfide bond. For example, a disulfide bond can link distinct scaffolds (for example, two isolated monomer scaffolds, an isolated monomer scaffold and a multimeric scaffold, or two multimeric scaffolds), a scaffold and a linker, a scaffold and an Fn domain, or a scaffold and an antibody or fragment thereof. In some embodiments, scaffolds of the invention comprise at least one non-naturally occurring intermolecular disulfide bond that links a scaffold and an isolated heterologous moiety, a scaffold and a heterologous moiety fused or conjugated to the same scaffold, or a scaffold and heterologous moiety fused or conjugated to a different scaffold.

In some embodiments, scaffolds of the invention comprise a disulfide bond that forms a multimeric scaffold of at least 2, at least 3, at least 4 or more scaffolds.

In another embodiment, scaffolds of the invention may comprise an elongation of the N and/or C terminal regions. In one embodiment, the scaffolds of the invention comprise an alteration to increase serum half-life, as described herein. In yet another embodiment, the scaffolds of the invention comprise an addition, deletion or substitution of at least one amino acid residue to stabilize the hydrophobic core of the scaffold.

In one embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the beta strands of the scaffold of the invention exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identity to the cognate beta strands of any one of SEQ ID NOs: 1-34, 54, 69, or 256, to the beta strands of any of the FnIII domains shown in FIG. 16, or to the beta strands of a protein domain recognized to contain the Interpro IPR008957 fibronectin type III domain signature as determined using the InterProScan program, or recognized to contain the Pfam PF00041 fibronectin type III domain signature as determined using Pfam_scan, HMMER, or any other program capable of comparing a protein sequence to a Hidden Markov model.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, or 131, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49, and the G beta strand comprises SEQ ID NO:52. In another specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:44, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:50, and the G beta strand comprises SEQ ID NO:53. In still another specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, or 131, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:51, and the G beta strand comprises SEQ ID NO:53.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, or 131, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:49, and the G beta strand consists of SEQ ID NO:52. In another specific embodiment, scaffolds of the invention consists at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:44, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:50, and the G beta strand consists of SEQ ID NO:53. In still another specific embodiment, scaffolds of the invention consists at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, or 131, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:51, and the G beta strand consists of SEQ ID NO:53.

In another embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, or 131, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:49, and the G beta strand consists essentially of SEQ ID NO:52. In another specific embodiment, scaffolds of the invention consists essentially at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:44, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:50, and the G beta strand consists essentially of SEQ ID NO:53. In a specific embodiment, scaffolds of the invention consists essentially of at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, or 131, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:51, and the G beta strand consists essentially of SEQ ID NO:53.

In another specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, or 131, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49, and the G beta strand comprises SEQ ID NO:52, wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand and the F beta strand (SEQ ID NOs: 45, or 131 and 49, respectively) may not be substituted.

In another specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, or 131, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:49, and the G beta strand consists of SEQ ID NO:52, wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand and the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be substituted.

In another specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, or 131, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:49, and the G beta strand consists essentially of SEQ ID NO:52, wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand and the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be substituted.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49, and the G beta strand comprises SEQ ID NO:52, the AB loop comprises SEQ ID NO:35, the CD loop comprises SEQ ID NO:37 and the EF loop comprises SEQ ID NO:39.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:49, and the G beta strand consists of SEQ ID NO:52, the AB loop consists of SEQ ID NO:35, the CD loop consists of SEQ ID NO:37 and the EF loop consists of SEQ ID NO:39.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:49, and the G beta strand consists essentially of SEQ ID NO:52, the AB loop consists essentially of SEQ ID NO:35, the CD loop consists essentially of SEQ ID NO:37 and the EF loop consists essentially of SEQ ID NO:39.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49, and the G beta strand comprises SEQ ID NO:52, the BC loop comprises SEQ ID NO:36, the DE loop comprises SEQ ID NO:38 and the FG loop comprises SEQ ID NO:40.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:49, and the G beta strand consists of SEQ ID NO:52, the BC loop consists of SEQ ID NO:36, the DE loop consists of SEQ ID NO:38 and the FG loop consists of SEQ ID NO:40.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:49, and the G beta strand consists essentially of SEQ ID NO:52, the BC loop consists essentially of SEQ ID NO:36, the DE loop consists essentially of SEQ ID NO:38 and the FG loop consists essentially of SEQ ID NO:40.

In another specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49, and the G beta strand comprises SEQ ID NO:52, wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand and the F beta strand (SEQ ID NOs: 45 and 49, respectively) may not be substituted.

In another specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:49, and the G beta strand consists of SEQ ID NO:52, wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand and the F beta strand (SEQ ID NOs: 45 and 49, respectively) may not be substituted.

In another specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:49, and the G beta strand consists essentially of SEQ ID NO:52, wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand and the F beta strand (SEQ ID NOs: 45 and 49, respectively) may not be substituted.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49, the G beta strand comprises SEQ ID NO:52, the AB loop comprises SEQ ID NO:35, the CD loop comprises SEQ ID NO:37, and the EF loop comprises SEQ ID NO:39 and, wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand and the F beta strand (SEQ ID NOs: 45 and 49, respectively) may not be substituted.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:49, the G beta strand consists of SEQ ID NO:52, the AB loop consists of SEQ ID NO:35, the CD loop consists of SEQ ID NO:37, and the EF loop consists of SEQ ID NO:39 and, wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand and the F beta strand (SEQ ID NOs: 45 and 49, respectively) may not be substituted.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:49, the G beta strand consists essentially of SEQ ID NO:52, the AB loop consists essentially of SEQ ID NO:35, the CD loop consists essentially of SEQ ID NO:37, and the EF loop consists essentially of SEQ ID NO:39 and, wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand and the F beta strand (SEQ ID NOs: 45 and 49, respectively) may not be substituted.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49, the G beta strand comprises SEQ ID NO:52, the BC loop comprises SEQ ID NO:36, the DE loop comprises SEQ ID NO:38, and the FG loop comprises SEQ ID NO:40 and, wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand and the F beta strand (SEQ ID NOs: 45 and 49, respectively) may not be substituted.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:49, the G beta strand consists of SEQ ID NO:52, the BC loop consists of SEQ ID NO:36, the DE loop consists of SEQ ID NO:38, and the FG loop consists of SEQ ID NO:40 and, wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand and the F beta strand (SEQ ID NOs: 45 and 49, respectively) may not be substituted.

In a specific embodiment, scaffolds of the invention comprise at least one non-naturally occurring intramolecular disulfide bond, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:49, the G beta strand consists essentially of SEQ ID NO:52, the BC loop consists essentially of SEQ ID NO:36, the DE loop consists essentially of SEQ ID NO:38, and the FG loop consists essentially of SEQ ID NO:40 and, wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine residues in the C beta strand and the E beta strand (SEQ ID NOs: 45 and 49, respectively) may not be substituted.

Enhanced Scaffold Stability: FG Loop Length

The inventors have discovered that the length of the FG loop plays a role in the stability of FnIII scaffolds. In particular, FnIII scaffolds comprising non-naturally occurring variant FG loops which are at least one amino acid shorter than that found in the FG loop of an FOI are shown to have enhanced stability. Accordingly, the present invention provides methods for obtaining a fibronectin type III (FnIII) scaffold variant having increased stability as compared to an FOI, comprising: engineering a variant of the FOI, wherein the FG loop of the variant comprises the deletion of at least 1 amino acid, and wherein the variant exhibits increased stability as compared to the FOI.

In certain embodiments, scaffolds of the invention comprise a non-naturally occurring variant FG loop which is at least one amino acid residue shorter than the FG loop of an FOI. For example, as defined herein the native FG loop of the third FnIII domain of human tenascin C comprises 10 amino acid residues. Accordingly, to identify an FnIII scaffold having improved stability using the third FnIII domain of human tenascin C as the FOI the FG loop would be reduced to 9 or fewer amino acid residues.

Although the sequence similarity between the amino acids sequences of the FnIII domains is generally low, the overall three dimensional structure is similar. Accordingly, using known techniques, such as sequence analysis and structure overlay, the FG loops of FnIII domains from multiple FOIs (e.g., FnIII domains from different species, different proteins, and different FnIII scaffolds that bind a target) may be determined (see for example FIG. 16. These loops can then be subjected to mutation to yield an FG loop that is at least one amino acid shorter than the FG loop from the FOI.

Thus, in one embodiment the instant invention encompasses FnIII scaffolds that comprise a non-naturally occurring variant FG loop which is at least one amino acid shorter than the FG loop of FOI regardless of what specific definition of the FG loop is used.

In a specific embodiment, the stability of an FOI is enhanced by deletion of at least one amino acid in the FG loop of the FOI. In another embodiment, the stability of an FOI is enhanced by deletion of at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 amino acids in the FG loop. It is specifically contemplated that the stabilized FOI may comprise at least one non-naturally occurring disulfide bond. In certain embodiments, the FOI comprised the non-naturally occurring intramolecular disulfide bond prior to being stabilized. In other embodiments, the stabilized FOI is further engineered to introduce at least one non-naturally occurring intramolecular disulfide bond.

In a specific embodiment, the invention provides a method of obtaining an FnIII scaffold variant having increased stability as compared to an FOI comprising engineering a variant of the FOI, wherein the FG loop of the variant comprises the deletion of at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 amino acids in the FG loop, wherein the variant exhibits an increased stability as compared to the FOI. In certain embodiments, the FnIII scaffold variant also comprises at least one loop, (i.e., AB, BC, CD, DE, EF, and/or FG) that has been randomized for length and/or sequence. It is specifically contemplated that the FnIII scaffold variant may comprise at least one non-naturally occurring disulfide bond. In certain embodiments, the FOI comprised the non-naturally occurring disulfide bond. In other embodiments, the FnIII variant is further engineered to introduce at least one non-naturally occurring disulfide bond.

In certain embodiments, the scaffold of the invention is an FnIII scaffold variant (i.e., a stabilized FOI) having increased stability as compared to an FOI, wherein the FnIII scaffold variant comprises an FG loop which is at least one, or at least two, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 amino acid residues shorter than the FG loop of the FOI, wherein the FnIII scaffold variant further comprises at least one amino acid substitution.

Stability Measurements

The increase in stability of the stabilized FnIII scaffolds of the invention, isolated or as part of a multimeric scaffold, can be readily measured by techniques well known in the art, such as thermal (T_(m)) and chaotropic denaturation (such as treatment with urea, or guanidine salts), protease treatment (such as treatment with thermolysin) or another art accepted methodology to determine protein stability. A comprehensive review of techniques used to measure protein stability can be found, for example in “Current Protocols in Molecular Biology” and “Current Protocols in Protein Science” by John Wiley and Sons. 2007.

In one embodiment the stabilized FnIII scaffolds of the invention exhibit an increase in stability of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more compared to the same FnIII scaffold prior to engineering (i.e., the FOI), as measured by thermal tolerance, resistance to chaotropic denaturation, protease treatment or another stability parameter well-known in the art.

The stability of a protein may be measured by the level of fluorescence exhibited by the protein under varying conditions. There is a positive correlation between the relative unfoldedness of a protein and a change in the internal fluorescence the protein exhibits under stress. Suitable protein stability assays to measure thermal unfolding characteristics include Differential Scanning calorimetry (DSC) and Circular Dichroism (CD). When the protein demonstrates a sizable shift in parameters measured by DSC or CD, it correlates to an unfolded structure. The temperature at which this shift is made is termed the melting temperature or (T_(m)).

In one embodiment, the stabilized scaffolds of the invention exhibit an increased melting temperature (T_(m)) of at least 1° C., at least 2° C., at least 3° C., at least 4° C., at least 5° C., at least 10° C., at least 15° C., at least 20° C., at least 25° C., at least 30° C., at least 35° C., at least 45° C., at least 50° C., at least 55° C., at least 60° C., at least 65° C., at least 70° C., at least 71° C., at least 72° C., at least 73° C., at least 74° C., at least 75° C., at least 76° C., at least 77° C., at least 78° C., at least 79° C., at least 80° C., at least 81° C., at least 82° C., at least 83° C., at least 84° C., at least 85° C., at least 85° C., at least 86° C., at least 87° C., at least 88° C., at least 89° C., at least 90° C., at least 91° C., at least 92° C., at least 93° C., at least 94° C., at least 94° C., at least, at least 95° C., at least 96° C., at least 97° C., at least 98° C., at least 100° C., at least 105° C., at least 110° C., or at least 120° C. as compared to the FOI under similar conditions.

In another embodiment, the stabilized FnIII scaffolds of the invention exhibit an increased melting temperature (T_(m)) of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more as compared to the FOI under similar conditions.

Another assay for protein stability involves exposing a protein to a chaotropic agent, such as urea or guanidine (for example, guanidine-HCl or guanidine isothiocynate) which acts to destabilize interactions within the protein. Upon exposing the protein to increasing levels of urea or guanidine, the relative intrinsic fluorescence is measured to assess a value in which 50% of the protein molecules are unfolded. This value is termed the C_(m) value and represents a benchmark value for protein stability. The higher the C_(m) value, the more stable the protein. In one embodiment, the stabilized FnIII scaffolds of the invention exhibit an increased C_(m) at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more as compared to the FOI as measured in a urea denaturation experiment under similar conditions. In another embodiment, the stabilized FnIII scaffolds of the invention exhibit an increased C_(m) at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more as compared to the FOI as measured in a guanidinium-HCl denaturation experiment under similar conditions.

Another assay used to assay protein stability is a protease resistance assay. In this assay, a relative level of protein stability is correlated with the resistance to protease degradation over time. The more resistant to protease treatment, the more stable the protein is. In one embodiment, the stabilized FnIII scaffolds of the invention exhibit increased stability by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more as compared to the FOI under similar conditions.

Multimeric Scaffolds

One aspect of the present invention provides multimeric scaffolds comprising at least two FnIII monomer scaffolds of the invention joined in tandem. Such multimeric scaffolds can be assembled in multiple formats. In some embodiments the monomer scaffolds are assembled in linear formats whereas in other embodiments the scaffolds are assembled in branched formats (see, e.g., FIG. 1). In a specific aspect, the invention provides multimeric scaffolds, wherein at least two FnIII scaffolds are connected in tandem via a peptide linker. In some embodiments, each FnIII scaffold in the multimeric scaffolds of the invention binds to a different target, thereby demonstrating multiple functions, and/or to the same target, thereby increasing the valency and/or avidity of target binding. In some embodiments, the increase in valency and/or avidity of target binding is accomplished when multiple scaffolds bind to the same target. In some embodiments, the increase in valency improves a specific action on the target, such as increasing the dimerization of a target protein.

In a specific embodiment, the multimeric scaffold of the invention comprises at least two FnIII monomer scaffolds of the invention connected in tandem, wherein each scaffold binds at least one target, and wherein each FnIII scaffold comprises a plurality of beta strands linked to a plurality of loop regions, wherein at least one loop is a non-naturally occurring variant of the cognate loop in an FOI, and wherein the beta strands of the FnIII scaffolds have at least 50% homology (i.e., sequence similarity) to the cognate beta strands of the FOI. In certain embodiments, each FnIII scaffold has at least 50% homology (i.e., sequence similarity) to the cognate beta strands of the same FOI. In a specific embodiment, each FnIII scaffold has at least 50% homology (i.e., sequence similarity) to the cognate beta strands of the wild type Tn3 scaffold (SEQ ID NO:1). It is specifically contemplated that each FnIII scaffold may have at least 50% homology (i.e., sequence similarity) to a different FOI. For example, a multimeric scaffold of the invention may comprise a first FnIII scaffold and a second FnIII scaffold, wherein the beta strands of the first FnIII scaffold have at least 50% homology (i.e., sequence similarity) to the cognate beta strands of the 14th FnIII domain of fibronectin (SEQ ID NOs:69), and wherein the beta strands of the second FnIII scaffold have at least 50% homology (i.e., sequence similarity) to the cognate beta strands of the wild type Tn3 scaffold (SEQ ID NO:1).

In some embodiments, a multimeric scaffold of the invention comprises at least two FnIII monomer scaffolds, wherein the FOI is the protein sequence corresponding to the third FnIII domain of human tenascin C. In a specific embodiment, the multimeric scaffold of the invention comprises at least two FnIII scaffolds, wherein the FOI is a wild type Tn3 scaffold. In other embodiments, the multimeric scaffold of the invention comprises at least two FnIII scaffolds, wherein the FOI is a protein sequence corresponding to an additional FnIII domain from human tenascin C. In other embodiments, the multimeric scaffold of the invention comprises at least two FnIII scaffolds, wherein the FOI is a protein sequence corresponding to an FnIII domain from another tenascin protein, or alternatively, a tenascin protein from another organism (such as, but not limited to, murine, porcine, bovine, or equine tenascins). In some embodiments, the multimeric scaffold of the invention comprises at least two FnIII scaffolds, wherein the beta strands of the FnIII scaffolds have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology (i.e., sequence similarity) to the cognate beta strands in any of: the 29^(th) FnIII domain from human tenascin XB (SEQ ID NO: 11), the 31^(st) FnIII domain from human tenascin XB (SEQ ID NO: 12), the 32^(nd) FnIII from human tenascin XB (SEQ ID NO: 13), the 3^(rd) FnIII domain of human fibronectin (SEQ ID NO: 6), the 6^(th) FnIII domain of human fibronectin (SEQ ID NO: 7), the 10^(th) FnIII domain of human fibronectin (e.g., SEQ ID NO: 5 and SEQ ID NO: 54), the 14^(th) FnIII domain of human fibronectin (e.g., SEQ ID NO: 69 and SEQ ID NO: 34), an FnIII domain from human growth hormone receptor (e.g., SEQ ID NO: 8 and SEQ ID NO: 15), an FnIII domain from beta common receptor (e.g., SEQ ID NO: 9), an FnIII from IL-5 receptor (e.g., SEQ ID NO: 10), an FnIII from PTPR-F (e.g., SEQ ID NO: 16 and SEQ ID NO: 17), or an FnIII domain from collagen type XIV (e.g., SEQ ID NO: 18).

In yet another embodiment the multimeric scaffold of the invention comprises at least two FnIII monomer scaffolds, wherein the FOI is a protein sequence corresponding to an FnIII domain from any organism. In other embodiments, the multimeric scaffold of the invention comprises at least two FnIII scaffolds, wherein a naturally occurring sequence corresponds to a predicted FnIII domain from a thermophilic or hyperthermophilic organism, for example, but not limited to Archaeoglobus fulgidus, Staphylothermus marinus, Sulfolobus acidocaldarius, Sulfolobus solfataricus, and Sulfolobus tokodaii. In some embodiments, the multimeric scaffold of the invention comprises at least two FnIII scaffolds, wherein the beta strands of the FnIII scaffolds have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology (i.e., sequence similarity) to the cognate beta strands in any of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33.

In one embodiment, the multimeric scaffold of the invention comprises at least two FnIII monomer scaffolds, wherein beta strands of the FnIII scaffolds have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology (sequence similarity) to the cognate beta strands in any one of the FnIII domains presented in FIG. 16, or to a protein domain recognized to contain the Interpro IPRO08957 fibronectin type III domain signature as determined using the InterProScan program, or recognized to contain the Pfam PF00041 fibronectin type III domain signature as determined using Pfam_scan, HMMER, or any other program capable of comparing a protein sequence to a Hidden Markov model.

Multimeric Tandem Scaffolds

In one embodiment, the multimeric scaffolds of the invention comprise two, three, four, five, six, eight or more FnIII monomer scaffolds of the invention. In some embodiments some of the FnIII monomer scaffolds are connected in tandem. In yet another embodiment, some of the FnIII monomer scaffolds are connected in tandem and some of the FnIII monomer scaffolds are not connected in tandem. In a specific embodiment, the multimeric scaffolds of the invention comprise two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or more scaffolds of the invention connected in tandem (see, e.g., FIG. 1 and FIG. 2).

In one embodiment, the multimeric scaffolds are generated through covalent binding between FnIII monomer scaffolds, for example, by directly linking the FnIII scaffolds, or by the inclusion of a linker, e.g., a peptide linker. In particular examples, covalently bonded scaffolds are generated by constructing fasion genes that encode the monomeric FnIII scaffolds or, alternatively, by engineering codons for cysteine residues into monomer FnIII scaffolds and allowing disulfide bond formation to occur between the expression products.

In one embodiment, the multimeric scaffolds of the invention comprise at least two FnIII scaffolds that are connected directly to each other without any additional intervening amino acids. In another embodiment, the multimeric scaffolds of the invention comprise at least two FnIII scaffolds that are connected in tandem via a linker, e.g., a peptide linker. In a specific embodiment, the multimeric scaffolds of the invention comprise at least two FnIII scaffolds that are connected in tandem via a peptide linker, wherein the peptide linker comprises 1 to about 1000, or 1 to about 500, or 1 to about 250, or 1 to about 100, or 1 to about 50, or 1 to about 25, amino acids. In a specific embodiment, the multimeric scaffolds of the invention comprise at least two FnIII scaffolds that are connected in tandem via a peptide linker, wherein the peptide linker comprises 1 to about 20, or 1 to about 15, or 1 to about 10, or 1 to about 5, amino acids.

In a specific embodiment, the multimeric scaffolds of the invention comprise at least two FnIII scaffolds that are connected in tandem via a linker, e.g., a peptide linker, wherein the linker is a functional moiety. The functional moiety will be selected based on the desired function and/or characteristics of the multimeric scaffold. For example, a functional moiety useful for purification (e.g., a histidine tag) may be used as a linker. Functional moieties useful as linkers include, but are not limited to, polyethylene glycol (PEG), a cytotoxic agent, a radionuclide, imaging agent, biotin, a dimerization domain (e.g. leucine zipper domain), human serum albumin (HSA) or an FcRn binding portion thereof, a domain or fragment of an antibody (e.g., antibody variable domain, a CH1 domain, a Ckappa domain, a Clambda domain, a CH2, or a CH3 domain), a single chain antibody, a domain antibody, an albumin binding domain, an IgG molecule, an enzyme, a ligand, a receptor, a binding peptide, a non-FnIII scaffold, an epitope tag, a recombinant polypeptide polymer, a cytokine, and the like. Specific peptide linkers and functional moieties which may be used as linkers are disclosed infra.

In some embodiments, the multimeric scaffolds of the invention comprise at least two FnIII scaffolds that are connected via one or more linkers, wherein the linkers interposed between each FnIII scaffold can be the same linkers or different linkers. In some embodiments, a linker can comprise multiple linkers, which can be the same linker or different linkers. In some embodiments, when a plurality of linkers are concatenated, some or all the linkers can be functional moieties.

Multimeric Scaffold Binding Stoichiometry

In some embodiments, the multimeric scaffolds of the invention comprise scaffolds that are specific for the same epitope. In other embodiments, multimeric scaffolds of the invention comprise scaffolds that are specific for different epitopes, which can be different epitopes on the same or different targets.

In a specific embodiment, the scaffolds of the multimeric scaffolds bind two or more different epitopes (e.g., non-overlapping epitopes) on the same target molecule. In another specific embodiment, the scaffolds of the multimeric scaffolds bind two or more different epitopes on the different target molecules. In yet another specific embodiment, the scaffolds of the multimeric scaffolds bind two or more different epitopes on the same target and additionally, bind at least one epitope on one or more different target molecules. In still another specific embodiment, the scaffolds of the multimeric scaffolds bind to the same epitope on a multimeric target molecule. In yet another embodiment, the scaffolds of the multimeric scaffolds bind to the same epitope on adjacent target molecules. In certain embodiments, the scaffolds of the multimeric scaffolds bind the same epitope on two or more copies of a target molecule on an adjacent cell surface. In some embodiments, the scaffolds of the multimeric scaffolds can bind to the same epitope or different epitopes in the same target or different targets with the same or different binding affinities and/or avidities.

In another embodiment, the monomer scaffolds in a multimeric scaffolds of the invention can bind to specific targets according to a specific binding pattern designed to achieve or enhance (e.g., synergistically) a desired effect. For example, the FnIII scaffolds in a linear multimeric scaffold can bind to a single target or to multiple targets according to a certain pattern, e.g., FnIII scaffolds in a 6 module linear multivalent scaffold can bind to two targets A and B according to an AAABBB pattern, an AABBAA pattern, an ABABAB pattern, an AAAABB pattern, etc.; to three targets according to an AABBCC pattern, an ABCABC pattern, and ABCCBA pattern, etc.; to four targets according to an ABCDDA patterns, ABCADA pattern, etc.; etc. In addition, when a multimeric scaffold of the invention comprises a plurality of engineered (e.g., disulfide engineered, loop engineered, or both disulfide and loop engineered) and non-engineered scaffolds, such monomeric scaffolds can be arranged according to a certain pattern to achieve or enhance a certain biological effect. Such combinations of monomeric scaffolds can be combinatorially assembled and subsequently evaluated using methods known in the art.

In some embodiments, multimeric scaffolds in branched constructs, e.g., multimeric scaffolds in an Fc fusion or antibody-like format, can also bind to a single target or to multiple targets according to a certain pattern. For instance, in certain embodiments a linear format scaffold fused to the IgG heavy chains in an antibody-like format scaffold can bind to a first target whereas a multivalent linear construct fused to the IgG light chains in an antibody-like format scaffold can bind to a second target. In another embodiment, linear format scaffolds fused to the IgG heavy chains of an antibody-like format scaffold can bind to a target with a certain affinity whereas the linear format scaffolds fused to the IgG light chains of an antibody-like format scaffold can bind to the same target with a different affinity. In some embodiments, the scaffolds fused to the chains in the left arm of the “Y” of an antibody can bind to a first target, whereas the scaffolds fused to the chains of the right of the “Y” of an antibody can bind to a second target.

Fusions

The invention further provides multimeric scaffolds comprising at least two FnIII monomer scaffolds, wherein at least one monomer scaffold may be fused to a heterologous moiety. In this context the heterologous moiety is not used to link the scaffolds as a spacer but may provide additional functionality to the multimeric scaffold of the invention. For example, in some embodiments, a multimeric scaffold that binds a target on the surface of a cell may be fused to a cytotoxic agent to facilitate target specific cell killing. Additional fusions are disclosed infra. In some embodiments, a heterologous moiety can function as a linker.

The present invention encompasses the use of scaffolds of the invention conjugated or fused to one or more heterologous moieties, including but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules. The present invention encompasses the use of scaffolds recombinantly fused or chemically conjugated to a heterologous protein or polypeptide or fragment thereof. Conjugation includes both covalent and non-covalent conjugation. In some embodiments, a scaffold of the invention can be fused or chemically conjugated to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 500, or at least 1000 amino acids) to generate fusion proteins.

The fusion or conjugation of a scaffold to one or more heterologous moieties can be direct, i.e., without a linker interposed between a scaffold and a heterologous moiety, or via one or more linker sequences described herein. In some embodiments, scaffolds can be used to target heterologous polypeptides to particular cell types, either in vitro or in vivo, by fusing or conjugating the scaffolds to antibodies specific for particular cell surface receptors in the target cells. Scaffolds fused or conjugated to heterologous polypeptides can also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., International Publication No. WO 93/21232; European Patent No. EP 439,095; Naramura et al. Immunol. Lett. 39:91-99, 1994; U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432, 1992; and Fell et al., J. Immunol. 146:2446-2452, 1991, which are incorporated by reference in their entireties.

In some embodiments, the scaffolds can be integrated with the human immune response by fusing or conjugating a scaffold with an immunoglobulin or domain thereof including, but not limited to, the constant region of an IgG (Fc), e.g., through the N or C-terminus. The Fc fusion molecule activates the complement component of the immune response and increases the therapeutic value of the protein scaffold. Similarly, a fusion between a scaffold and a complement protein, such as CIq, can be used to target cells. A fusion between scaffold and a toxin can be used to specifically destroy cells that carry a particular antigen as described herein.

Various publications describe methods for obtaining physiologically active molecules whose half-lives are modified by introducing an FcRn-binding polypeptide into the molecules (see, e.g., WO 97/43316; U.S. Pat. No. 5,869,046; U.S. Pat. No. 5,747,035; WO 96/32478; and WO 91/14438), by fusing the molecules with antibodies whose FcRn-binding affinities are preserved but affinities for other Fc receptors have been greatly reduced (See, e.g., WO 99/43713), or by fusing the molecules with FcRn binding domains of antibodies (see, e.g., WO 00/09560; U.S. Pat. No. 4,703,039). Specific techniques and methods of increasing half-life of physiologically active molecules can also be found in U.S. Pat. No. 7,083,784. Specifically, it is contemplated that the scaffolds of the invention can be fused to an Fc region from an IgG, wherein the Fc region comprises amino acid residue mutations M252Y/S254T/T256E or H433K/N434F/Y436H, wherein amino acid positions are designated according to the Kabat numbering schema. In some embodiments, the half life of a multimeric scaffold of the invention is increased by genetically fusing the multimeric scaffold with an intrinsically unstructured recombinant polypeptide (e.g., an XTEN™ polypeptide) or by conjugation with polyethylene glycol (PEG).

In some embodiments, the scaffolds of the invention can be fused with molecules that increase or extend in vivo or serum half life. In some embodiments, the scaffolds of the invention are fused or conjugated with albumin, such as human serum albumin (HSA), a neonatal Fc receptor (FcRn) binding fragment thereof, polyethylene glycol (PEG), polysaccharides, immunoglobulin molecules (IgG) or fragments thereof, complement, hemoglobin, a binding peptide, lipoproteins and other factors to increase its half-life in the bloodstream and/or its tissue penetration. Any of these fusions may be generated by standard techniques, for example, by expression of the fusion protein from a recombinant fusion gene constructed using publicly available gene sequences.

Moreover, the scaffolds of the invention can be fused to marker sequences, such as a peptide to facilitate purification. In some embodiments, the marker amino acid sequence is a poly-histidine peptide (His-tag), e.g., a octa-histidine-tag (His-8-tag) or hexa-histidine-tag (His-6-tag) such as the tag provided in a pQE expression vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among other vectors, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989, for instance, poly-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, a hemagglutinin (“HA”) tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (see, e.g., Wilson et al., Cell 37:767, 1984), a FLAG tag, a Strep-tag, a myc-tag, a V5 tag, a GFP-tag, an AU1-tag, an AU5-tag, an ECS-tag, a GST-tag, or an OLLAS tag.

Additional fusion proteins comprising scaffolds of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to alter the activities of scaffolds of the invention (e.g., scaffolds with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33, 1997; Harayama, Trends Biotechnol. 16(2):76-82, 1998; Hansson, et al., J. Mol. Biol. 287:265-76, 1999; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313 (each of these patents and publications is hereby incorporated by reference in its entirety). Scaffolds, or the encoded scaffolds thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. One or more portions of a polynucleotide encoding a scaffold, which bind to a specific target may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Antibody-Like Multimeric Scaffolds

In some embodiments, the multimeric scaffold of the invention comprise at least two FnIII, wherein at least one scaffold is fused to a domain or fragment of an antibody (e.g., an IgG), including but not limited to an intact antibody, an antibody variable domain, a CH1 domain, a Ckappa domain, a Clambda domain, an Fc domain, a CH2, or a CH3 domain.

In some embodiments, scaffolds of the invention can be fused to a domain or fragment of an antibody. The domain or fragment of an antibody further enhances the avidity and/or affinity of the multimeric scaffold by providing, similarly to the Fc domain described below, a dimerization or multimerization domain which facilitates the formation of multimeric scaffolds of the invention.

In some embodiments, only one multimeric tandem scaffold comprising two FnIII domains is conjugated or fused to a domain or fragment of an antibody. For instance, a single multimeric tandem scaffold can be fused to the N-terminus of a polypeptide of a domain or fragment of an antibody (e.g., a heavy chain or a light chain of an antibody). In some embodiments, multivalent scaffolds are created by fusing or conjugating one or more FnIII scaffolds to the N-terminus and/or the C-terminus a polypeptide of a domain or fragment of an antibody (e.g., a heavy chain and/or a light chain of an antibody. In some embodiments, some or all the scaffolds fused to a domain or fragment of an antibody are identical. In some other embodiments, some or all the scaffolds fused to a domain or fragment of an antibody are different.

In some embodiments, the scaffolds of the invention used to generate an antibody-like multivalent scaffold can contain the same number of FnIII modules. In other embodiments, the scaffolds of the invention used to generate an antibody-like multivalent scaffold can contain a different number of FnIII modules. For example, a tetravalent FnIII scaffold can be formed, e.g., by fusing a linear format tetravalent FnIII scaffold to a single position, or by fusing one FnIII monomer scaffold in one position and a trimeric linear format FnIII scaffold to another position, or by fusing two dimeric FnIII linear format scaffolds to two different positions, or by fusing 4 FnIII monomer scaffolds, each one to a single position.

In a specific embodiment, multimeric FnIII scaffolds of the invention comprise four multimeric linear scaffolds fused to a domain or fragment of an antibody wherein each multimeric linear scaffold comprises two FnIII monomer scaffolds that are connected in tandem via a linker (FIG. 1). In other embodiments, multimeric FnIII scaffolds of the invention comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven or at least eight monomeric or multimeric FnIII scaffolds of the invention fused to a domain or fragment of an antibody.

In one specific embodiment, a tetravalent FnIII scaffold can be generated by fusing one scaffold to the N-terminus of each of the light chains and heavy chains of a domain or fragment of an antibody (see, e.g., A9 construct in FIG. 2).

An antibody-like format multivalent FnIII scaffold can be generated by fusing any combination of scaffolds of the invention to a domain or fragment of an antibody or modified antibody. Examples of modified antibodies include domain deleted antibodies, minibodies (see, e.g., U.S. Pat. No. 5,837,821), tetravalent minibodies, tetravalent antibodies (see, e.g., Coloma & Morrison, Nature Biotechnol. 15:159-163, 1997; PCT Publication No. WO 95/09917), thermally stabilized antibodies, humanized antibodies, etc.

Each of the linear scaffolds of the invention used to generate an antibody-like multivalent scaffold according to FIG. 1 can contain the same linkers and linker distributions, or different linkers and different linker distributions.

Fc-Fusion Multimeric Scaffolds

In some embodiments, a multimeric scaffold of the invention comprises a plurality of monomeric or multimeric scaffolds connected to an Fc domain. The fusion of a multimeric scaffold of the invention to an antibody fragment comprising an Fc domain further enhances the avidity and/or activity of the multimeric FnIII scaffold by providing a dimerization domain which facilitates the formation of dimers of the multimeric FnIII scaffolds.

In some embodiments, only one multimeric FnIII scaffold is fused to an Fc domain. In a specific embodiment, multimeric scaffolds of the invention comprise two multimeric FnIII scaffolds fused to an Fc domain wherein each multimeric FnIII scaffold comprises two or more FnIII scaffolds that are connected via one or more linkers (FIG. 1). In one specific embodiment, the multimeric FnIII scaffolds fused to the Fc domain are linear format scaffolds.

In one specific embodiment, two linear format FnIII scaffolds comprising two FnIII domains in tandem are fused to an Fc domain to yield a multimeric scaffold with a valency of 4 (see, e.g., A7 construct in FIG. 2). In another specific embodiment, two linear format scaffolds, each one of them comprising four FnIII monomer scaffolds are fused to an Fc domain to yield an FnIII multimeric scaffold with a valency of 8 (see, e.g., A8 construct in FIG. 2).

In some embodiments, the FnIII scaffolds fused to the Fc domain comprise the same number of FnIII modules. In some embodiments, the FnIII scaffolds fused to the Fc domain comprise a different number of FnIII modules. In some embodiments, the FnIII scaffolds fused to the Fc domain comprise the same linkers. In other embodiments, the FnIII scaffolds fused to the Fc domain comprise different linkers.

In some embodiments, different multimeric FnIII scaffolds of the invention can be dimerized by the use of Fc domain mutations which favor the formation of heterodimers. See, for example, WO96/27011 which describes a method, in which one or more small amino acid side chains from the interface of a first Fc domain are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of a second Fc domain by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

It is known that variants of the Fc region (e.g., amino acid substitutions and/or additions and/or deletions) enhance or diminish effector function of the antibody (see, e.g., U.S. Pat. Nos. 5,624,821; 5,885,573; 6,538,124; 7,317,091; 5,648,260; 6,538,124; International Publications Nos. WO 03/074679; WO 04/029207; WO 04/099249; WO 99/58572; US Publication No. 2006/0134105; 2004/0132101; 2006/0008883) and can alter the pharmacokinetic properties (e.g. half-life) of the antibody (see, U.S. Pat. Nos. 6,277,375 and 7,083,784). Thus, in certain embodiments, the multispecific FnIII scaffolds of the invention comprise Fc domain(s) that comprise an altered Fc region in which one or more alterations have been made in the Fc region in order to change functional and/or pharmacokinetic properties of the multimeric FnIII scaffolds.

It is also known that the glycosylation of the Fc region can be modified to increase or decrease effector function and/or anti-inflammatory activity (see, e.g., Umana et al., Nat. Biotechnol. 17:176-180, 1999; Davies et al. Biotechnol. Bioeng. 74:288-294, 2001; Shields et al., J. Biol. Chem. 277:26733-26740, 2002; Shinkawa et al., J. Biol. Chem. 278:3466-3473, 2003; U.S. Pat. Nos. 6,602,684; 6,946,292; 7,064,191; 7,214,775; 7,393,683; 7,425,446; 7,504,256; U.S. Publication. Nos. 2003/0157108; 2003/0003097; 2009/0010921; Potillegent™ technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); Keneko et al., Science 313:670-673, 2006; Scallon et al., Mol. Immuno. 44(7):1524-34, 2007). Accordingly, in one embodiment the Fc regions of the multimeric FnIII scaffolds of the invention comprise altered glycosylation of amino acid residues in order to change cytotoxic and/or anti-inflammatory properties of the multimeric scaffolds.

Multimeric Scaffold Topologies

One skilled in the art would appreciate that multimeric scaffolds discussed above, in FIG. 1 and FIG. 2, and throughout the specification are just illustrative examples. The construct topologies or formats shown in FIG. 1 and FIG. 2 illustrate that in some embodiments the scaffolds of the invention are fused to the N-termini of the constituent polypeptides of Fc domains and antibodies. The scaffolds of the invention can be fused to the C-terminus of the Fc domains, antibody light chains, and antibody heavy chains in any suitable spatial arrangement. For example, an some embodiments a tetravalent scaffold can be created by fusing an FnIII monomer scaffold to the N-terminus of each heavy chain and an FnIII monomer scaffold to the C-terminus domain of each light chain of an antibody, by fusing an FnIII monomer scaffold to the N-terminus of each light chain and an FnIII monomer scaffold to the C-terminus of each heavy chain of an antibody, or by fusing an FnIII monomer scaffold to the N-terminus of each heavy chain and an FnIII monomer scaffold to the N-terminus of each light chain of an antibody. Monomeric and/or multimeric FnIII scaffolds can be fused to full length heavy and/or light chains comprising both variable regions and constant regions. Alternatively, monomeric and/or multimeric FnIII scaffolds can be fused to truncated heavy and/or light chains comprising only constant regions (e.g., as in the A9 construct shown in FIG. 2).

Multimeric scaffolds can be created by using the formats shown in FIG. 1 as building blocks. For example, the antibody-like and Fc fusion formats are combinations comprising more simple linear format modules. Accordingly, in some embodiments more complex multimeric scaffolds formats can be created by combining the building blocks shown in FIG. 1.

FIGS. 1 and 2 also illustrate that in some embodiments the multimeric scaffolds of the invention can be linear or branched and exhibit different levels of branching. For example, the Fc format provides an example of first order branched format, whereas the antibody-like format provides an example of a second-order branched format. Higher order branched constructs can be obtained by replacing the linear format building blocks in the antibody-like format or the Fc fusion format with Fc fusion format building blocks or antibody-like building blocks, and connect them to either the C-termini or N-termini of the constituent polypeptides of the Fc or antibody.

FIGS. 1 and 2, and TABLE 1 illustrate the fact that in some embodiments the linkers in a multimeric scaffold can be structurally and functionally diverse and can provide a plurality of attachment points. For example, all the FnIII modules in the A4 and A5 constructs are connected by (Gly-4-Ser)₃Ala linkers, except for the 4th and 5th FnIII modules, which are connected by a (Gly₄-Ser)-2-Gly-Thr-Gly-Ser-Ala-Met-Ala-Ser-(Gly₄-Ser)₁-Ala linker. For example, in the A7 construct, the first and second FnIII domains and the third and fourth FnIII domain are connected by (Gly₄-Ser)₃Ala linkers, whereas the second and third FnIII domains are connected by an Fc domain as a functional moiety linker.

The Fc fusion shown in FIG. 1 exemplifies that in some embodiments monomeric or multimeric FnIII scaffolds can be fused to the N-termini of the polypeptides of the functional moiety linker. In some embodiments, monomeric or multimeric FnIII scaffolds of the invention can readily be fused to the C-terminus of the Fc domain in an Fc fusion format construct.

Similarly, the antibody or modified antibody in an antibody-like format construct is also a functional moiety linker. In this case, instead of two attachment points as in a (Gly₄-Ser)₃Ala or (Gly₄-Ser)₂Gly-Thr-Gly-Ser-Ala-Met-Ala-Ser-(Gly₄-Ser)₁-Ala, or four possible attachment points as in the Fc domain case, the antibody shown in the antibody-like example of FIG. 1 provides 6 possible attachment points. The antibody-like format shown in FIG. 1 exemplifies that in some embodiments only the N-terminal attachment points in the functional moiety linker are occupied by FnIII domains of the invention. In an antibody-like format construct some or all that scaffolds of the invention can readily be fused to the C-termini of the heavy chains and the light chains of an antibody or modified antibody domain. Other fusion stoichiometries can be applied, i.e., one, two, three, four, five, six, seven, eight, or more scaffolds of the invention can be fused to an antibody or modified antibody.

FIGS. 1 and 2 also illustrate that in some embodiments multimeric FnIII scaffolds can be generated by combining other FnIII multimeric scaffolds. For example, the Fc format A6, A7, and A8 scaffolds of FIG. 2 are homodimeric FnIII scaffolds wherein the multimeric scaffold is formed by two polypeptide chains, each one comprising a linear format FnIII scaffold fused to an Fc domain, which in turn are connected via intermolecular disulfide bonds. The antibody-like format scaffold of FIGS. 1 and 2 exemplifies a heterotetrameric FnIII scaffold wherein 4 polypeptides corresponding to two different types of scaffolds (2 FnIII scaffolds formed by fusing an FnIII monomer scaffold to an IgG light chain constant region, and 2 FnIII scaffolds formed by fusing an FnIII monomer scaffold to an CH1-hinge-region-Fc region of an IgG) are connected via intermolecular disulfide bonds.

Generation of Scaffolds of the Invention

The FnIII scaffolds described herein may be used in any technique for evolving new or improved target binding proteins. In one particular example, the target is immobilized on a solid support, such as a column resin or microtiter plate well, and the target contacted with a library of candidate scaffold-based binding proteins. Such a library may consist of clones constructed from an FnIII domain, including without limitation the Tn3 module, through randomization of the sequence and/or the length of the CDR-like loops. In one embodiment, the library may be a phage, phagemid, virus, bacterial, yeast, or mammalian cell display or a ribosome display library. If desired, this library may be an RNA-protein fusion library generated, for example, by the techniques described in Szostak et al., U.S. Pat. Nos. 6,258,558; 6,261,804; 5,643,768; and 5,658,754. Alternatively, it may be a DNA-protein library (for example, as described in PCT Publ. No. WO 2000/032823).

In this regard, bacteriophage (phage) display is one well known technique which allows one to screen large oligopeptide libraries to identify member(s) of those libraries which are capable of specifically binding to a target. Phage display is a technique by which variant polypeptides are displayed as fusion proteins to the coat protein on the surface of bacteriophage particles (Scott, J. K. and Smith, G. P. (1990) Science 249: 386). The utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have been used for screening millions of polypeptides or oligopeptides for ones with specific binding properties (Smith, G. P. (1991) Current Opin. Biotechnol., 2:668). Sorting phage libraries of random mutants requires a strategy for constructing and propagating a large number of variants, a procedure for affinity purification using the target receptor, and a means of evaluating the results of binding enrichments (see for example, U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,689, and 5,663,143).

Although most phage display methods have used filamentous phage, lambdoid phage display systems (WO 95/34683; U.S. Pat. No. 5,627,024), T4 phage display systems (Ren et al., Gene, 215: 439 (1998); Zhu et al., Cancer Research, 58(15): 3209-3214 (1998); Jiang et al., Infection & Immunity, 65(11): 4770-4777 (1997); Ren et al., Gene, 195(2):303-311 (1997); Ren, Protein Sci., 5: 1833 (1996); Efimov et al., Virus Genes, 10: 173 (1995)) and T7 phage display systems (Smith and Scott, Methods in Enzymology, 217: 228-257 (1993); U.S. Pat. No. 5,766,905) are also known.

Many other improvements and variations of the basic phage display concept have now been developed. These improvements enhance the ability of display systems to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential of screening these proteins for desired properties. Combinatorial reaction devices for phage display reactions have been developed (WO 98/14277) and phage display libraries have been used to analyze and control bimolecular interactions (WO 98/20169; WO 98/20159) and properties of constrained helical peptides (WO 98/20036). WO 97/35196 describes a method of isolating an affinity ligand in which a phage display library is contacted with one solution in which the ligand will bind to a target molecule and a second solution in which the affinity ligand will not bind to the target molecule, to selectively isolate binding ligands. WO 97/46251 describes a method of biopanning a random phage display library with an affinity purified antibody and then isolating binding phage, followed by a micropanning process using microplate wells to isolate high affinity binding phage. The use of Staphylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) Mol. Biotech., 9:187). WO 97/47314 describes the use of substrate subtraction libraries to distinguish enzyme specificities using a combinatorial library which may be a phage display library. A method for selecting enzymes suitable for use in detergents using phage display is described in WO 97/09446. Additional methods of selecting specific binding proteins are described in U.S. Pat. Nos. 5,498,538, 5,432,018, and WO 98/15833.

Methods of generating peptide libraries and screening these libraries are also disclosed in U.S. Pat. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and 5,723,323.

A bioinformatics approach may be employed to determine the loop length and diversity preferences of naturally occurring FnIII domains. Using this analysis, the preferences for loop length and sequence diversity may be employed to develop a “restricted randomization” approach. In this restricted randomization, the relative loop length and sequence preferences are incorporated into the development of a library strategy. Integrating the loop length and sequence diversity analysis into library development results in a restricted randomization (i.e. certain positions within the randomized loop are limited in which amino acid could reside in that position).

The invention also provides recombinant libraries (hereinafter referred to as “libraries of the invention”) comprising diverse populations of non-naturally occurring FnIII scaffolds of the invention. In one embodiment, the libraries of the invention comprise non-naturally occurring FnIII scaffolds comprising, a plurality of beta strand domains linked to a plurality of loop regions, wherein one or more of said loops vary by deletion, substitution or addition by at least one amino acid from the cognate loops in an FOI, and wherein the beta strands of the FnIII scaffold have at least 50% homology (i.e., sequence similarity) to the cognate beta strand sequences of the FOI. Non-limiting examples of FOI sequences useful for the generation of recombinant libraries are provided in TABLE 1 and in FIG. 16.

In some embodiments, libraries of the invention comprise non-naturally occurring FnIII scaffolds, wherein the FOI is the protein sequence corresponding to the third FnIII domain of human tenascin C. In some embodiments, libraries of the invention comprise non-naturally occurring FnIII scaffolds, wherein the FOI is the protein sequence corresponding to the tenth FnIII domain of human fibronectin. In some embodiments, libraries of the invention comprise non-naturally occurring FnIII scaffolds, wherein the FOI is the protein sequence corresponding to the fourteenth FnIII domain of human fibronectin.

In a specific embodiment, the libraries of the invention comprise non-naturally occurring FnIII scaffolds, wherein the FOI is a wild type Tn3 scaffold. In other embodiments, libraries of the invention comprise non-naturally occurring FnIII scaffolds, wherein the FOI is a protein sequence corresponding to an additional FnIII domain from human tenascin C. In other embodiments, libraries of the invention comprise non-naturally occurring FnIII scaffolds, wherein the FOI is a protein sequence corresponding to a FnIII domain from another tenascin protein, or alternatively, a tenascin protein from another organism (such as, but not limited to, murine, porcine, bovine, or equine tenascins).

In yet another embodiment, libraries of the invention comprise non-naturally occurring FnIII scaffolds, wherein the FOI is a protein sequence corresponding to a FnIII domain from any organism. In other embodiments, libraries of the invention comprise non-naturally occurring FnIII scaffolds, wherein the naturally occurring sequence corresponds to a predicted FnIII domain from a thermophilic or hyperthermophilic organism. For example, the hyperthermophilic organism can be a hyperthermophilic archaea such as Archaeoglobus fulgidus, Staphylothermus marinus, Sulfolobus acidocaldarius, Sulfolobus solfataricus, and Sulfolobus tokodaii.

In some embodiments, the libraries of the invention comprise FnIII scaffolds, wherein the beta strands of the FnIII scaffold have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homology (sequence similarity) to the cognate beta strain domain in any of SEQ ID NOs: 1-34, 54, 69 or those presented in FIG. 16, or to the beta strands of a domain recognized to contain the Pfam PF00041 fibronectin type III domain signature as determined using Pfam_scan, HMMER, or any other program capable of comparing a protein sequence to a Hidden Markov model.

As detailed above, the loops connecting the various beta strands of the scaffolds may be randomized for length and/or sequence diversity. In one embodiment, the libraries of the invention comprise FnIII scaffolds having at least one loop that is randomized for length and/or sequence diversity. In one embodiment, at least one, at least two, at least three, at least four, at least five or at least six loops of the FnIII scaffolds are randomized for length and/or sequence diversity. In one embodiment, at least one loop is kept constant while at least one additional loop is randomized for length and/or sequence diversity. In another embodiment, at least one, at least two, or all three of loops AB, CD, and EF are kept constant while at least one, at least two, or all three of loops BC, DE, and FG are randomized for length or sequence diversity. In another embodiment, at least one, at least two, or at least all three of loops AB, CD, and EF are randomized while at least one, at least two, or all three of loops BC, DE, and FG are randomized for length and/or sequence diversity.

As detailed above, it has been surprisingly found that FG loops which are at least one amino acid shorter than that found in the FG loop of an FOI are shown to have enhanced stability. Accordingly the present invention provides libraries of the invention comprising FnIII scaffolds, wherein at least one loop is randomized for length and/or sequence diversity, except that length of the FG loops are held to be at least one amino acid shorter than the cognate FG loop of an FOI. For example, as defined in FIG. 16, the native FG loop of the third FnIII domain of human tenascin C comprises 10 amino acid residues, accordingly, where the third FnIII domain of human tenascin C is the FOI the FG loop would be held to 9 amino acid residues or less although the sequence of the FG loop may be randomized.

In some embodiments, the libraries of the invention comprise FnIII scaffolds, wherein each scaffold comprises seven beta strands designated A, B, C, D, E, F, and G linked to six loop regions, wherein a loop region connects each beta strand and is designated AB, BC, CD, DE, EF, and FG; and wherein at least one loop is a non-naturally occurring variant of a FOI loop, and wherein the FG loop is at least one amino acid shorter than the cognate FG loop in the FOI.

In one embodiment, libraries of the invention comprise FnIII scaffold, wherein the amino acid sequence of one or more loops (i.e., AB, BC, CD, DE, EF, FG) has been randomized for length and/or sequence diversity, except that the length of the FG loops are held to be at least one, or at least two, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 amino acid residue shorter than the cognate FG loop of an FOI.

In certain embodiments, the libraries of the invention comprise FnIII scaffolds, wherein each beta strand has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more homology (sequence similarity) to the cognate beta strands of any one of SEQ ID NOs: 1-34, 54, or 69, to the beta strands of any of the FnIII domains shown in FIG. 16, or to the beta strands of a domain recognized to contain the Pfam PF00041 fibronectin type III domain signature as determined using Pfam_scan, HMMER, or any other program capable of comparing a protein sequence to a Hidden Markov model.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand comprises SEQ ID NO: 42, the B beta strand comprises SEQ ID NO: 43, the C beta strand comprises SEQ ID NO: 45, or 131, the D beta strand comprises SEQ ID NO: 46, the E beta strand comprises SEQ ID NO: 47, the F beta strand comprises SEQ ID NO: 49, and the G beta strand comprises SEQ ID NO: 52. In another specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:44, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:50, and the G beta strand comprises SEQ ID NO:53. In still another specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, or 131, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:51, and the G beta strand comprises SEQ ID NO:53.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand consists of SEQ ID NO: 42, the B beta strand consists of SEQ ID NO: 43, the C beta strand consists of SEQ ID NO: 45, or 131, the D beta strand consists of SEQ ID NO: 46, the E beta strand consists of SEQ ID NO: 47, the F beta strand consists of SEQ ID NO: 49, and the G beta strand consists of SEQ ID NO: 52. In another specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:44, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:50, and the G beta strand consists of SEQ ID NO:53. In still another specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, or 131, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:51, and the G beta strand consists of SEQ ID NO:53.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand consists essentially of SEQ ID NO: 42, the B beta strand consists essentially of SEQ ID NO: 43, the C beta strand consists essentially of SEQ ID NO: 45, or 131, the D beta strand consists essentially of SEQ ID NO: 46, the E beta strand consists essentially of SEQ ID NO: 47, the F beta strand consists essentially of SEQ ID NO: 49, and the G beta strand consists essentially of SEQ ID NO: 52. In another specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:44, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:50, and the G beta strand consists essentially of SEQ ID NO:53. In still another specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, or 131, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:51, and the G beta strand consists essentially of SEQ ID NO:53.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49, and the G beta strand comprises SEQ ID NO:52, the AB loop comprises SEQ ID NO:35, the CD loop comprises SEQ ID NO:37 and the EF loop comprises SEQ ID NO:39.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:49, and the G beta strand consists of SEQ ID NO:52, the AB loop consists of SEQ ID NO:35, the CD loop consists of SEQ ID NO:37 and the EF loop consists of SEQ ID NO:39.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:49, and the G beta strand consists essentially of SEQ ID NO:52, the AB loop consists essentially of SEQ ID NO:35, the CD loop consists essentially of SEQ ID NO:37 and the EF loop consists essentially of SEQ ID NO:39.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49, and the G beta strand comprises SEQ ID NO:52, the BC loop comprises SEQ ID NO:36, the DE loop comprises SEQ ID NO:38 and the FG loop comprises SEQ ID NO:40.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:49, and the G beta strand consists of SEQ ID NO:52, the BC loop consists of SEQ ID NO:36, the DE loop consists of SEQ ID NO:38 and the FG loop consists of SEQ ID NO:40.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, or 131, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:49, and the G beta strand consists essentially of SEQ ID NO:52, the BC loop consists essentially of SEQ ID NO:36, the DE loop consists essentially of SEQ ID NO:38 and the FG loop consists essentially of SEQ ID NO:40.

In another specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand comprises SEQ ID NO: 42, the B beta strand comprises SEQ ID NO: 43, the C beta strand comprises SEQ ID NO: 45, or 131, the D beta strand comprises SEQ ID NO: 46, the E beta strand comprises SEQ ID NO: 47, the F beta strand comprises SEQ ID NO: 49, and beta strand G comprises SEQ ID NO: 52, and wherein one or more of the beta strands comprise at least one amino acid substitution except that the cysteine in the C beta strand and the cysteine in the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be substituted.

In another specific embodiment, the libraries of the invention comprise FnIll scaffolds, wherein the A beta strand consists of SEQ ID NO: 42, the B beta strand consists of SEQ ID NO: 43, the C beta strand consists of SEQ ID NO: 45, or 131, the D beta strand consists of SEQ ID NO: 46, the E beta strand consists of SEQ ID NO: 47, the F beta strand consists of SEQ ID NO: 49, and beta strand G consists of SEQ ID NO: 52, and wherein one or more of the beta strands comprise at least one amino acid substitution except that the cysteine in the C beta strand and the cysteine in the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be substituted.

In another specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand consists essentially of SEQ ID NO: 42, the B beta strand consists essentially of SEQ ID NO: 43, the C beta strand consists essentially of SEQ ID NO: 45, or 131, the D beta strand consists essentially of SEQ ID NO: 46, the E beta strand consists essentially of SEQ ID NO: 47, the F beta strand consists essentially of SEQ ID NO: 49, and beta strand G consists essentially of SEQ ID NO: 52, and wherein one or more of the beta strands comprise at least one amino acid substitution except that the cysteine in the C beta strand and the cysteine in the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be substituted.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, or 131, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49, the G beta strand comprises SEQ ID NO:52, the AB loop comprises SEQ ID NO:35, the CD loop comprises SEQ ID NO:37, and the EF loop comprises SEQ ID NO:39 and, wherein one or more of the beta strands comprise at least one amino acid substitution except that the cysteine in the C beta and the cysteine in the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be substituted.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, or 131, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:49, the G beta strand consists of SEQ ID NO:52, the AB loop consists of SEQ ID NO:35, the CD loop consists of SEQ ID NO:37, and the EF loop consists of SEQ ID NO:39 and, wherein one or more of the beta strands comprise at least one amino acid substitution except that the cysteine in the C beta and the cysteine in the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be substituted.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, or 131, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:49, the G beta strand consists essentially of SEQ ID NO:52, the AB loop consists essentially of SEQ ID NO:35, the CD loop consists essentially of SEQ ID NO:37, and the EF loop consists essentially of SEQ ID NO:39 and, wherein one or more of the beta strands comprise at least one amino acid substitution except that the cysteine in the C beta and the cysteine in the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be substituted.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand domain comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, or 131, the D beta strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49, the G beta strand comprises SEQ ID NO:52, the BC loop comprises SEQ ID NO:36, the DE loop comprises SEQ ID NO:38, and the FG loop comprises SEQ ID NO:40 and, wherein one or more of the beta strands comprise at least one amino acid substitution except that the cysteine in the C beta strand and the cysteine in the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be substituted.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand domain consists of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta strand consists of SEQ ID NO:45, or 131, the D beta strand consists of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta strand consists of SEQ ID NO:49, the G beta strand consists of SEQ ID NO:52, the BC loop consists of SEQ ID NO:36, the DE loop consists of SEQ ID NO:38, and the FG loop consists of SEQ ID NO:40 and, wherein one or more of the beta strands comprise at least one amino acid substitution except that the cysteine in the C beta strand and the cysteine in the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be substituted.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the A beta strand domain consists essentially of SEQ ID NO:42, the B beta strand consists essentially of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID NO:45, or 131, the D beta strand consists essentially of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID NO:49, the G beta strand consists essentially of SEQ ID NO:52, the BC loop consists essentially of SEQ ID NO:36, the DE loop consists essentially of SEQ ID NO:38, and the FG loop consists essentially of SEQ ID NO:40 and, wherein one or more of the beta strands comprise at least one amino acid substitution except that the cysteine in the C beta strand and the cysteine in the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be substituted.

As detailed above, one or more residues within a loop may be held constant while other residues are randomized for length and/or sequence diversity. Optionally or alternatively, one or more residues within a loop may be held to a predetermined and limited number of different amino acids while other residues are randomized for length and/or sequence diversity. Accordingly, libraries of the invention comprise FnIII scaffolds that may comprise one or more loops having a degenerate consensus sequence and/or one or more invariant amino acid residues. In one embodiment, the libraries of the invention comprise FnIII scaffolds having AB loops which are randomized with the following consensus sequence: K-X-X-X-X-X-a, wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, and wherein (a) represents serine, threonine, alanine, or glycine. In another embodiment, the libraries of the invention comprise FnIII scaffolds having AB loops which are randomized with the following consensus sequence: K-X-X-X-X-X-X-X-a, wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, and wherein (a) represents serine, threonine, alanine, or glycine.

In another embodiment, the libraries of the invention comprise FnIII scaffolds having BC loops which are randomized with the following consensus sequence: S-X-a-X-b-X-X-X-G, wherein X represents any amino acid, wherein (a) represents proline or alanine and wherein (b) represents alanine or glycine. In another embodiment, the libraries of the invention comprise FnIII scaffolds having BC loops which are randomized with the following consensus sequence: S-P-c-X-X-X-X-X-X-T-G, wherein X represents any amino acid and wherein (c) represents proline, serine or glycine. In still another embodiment, the libraries of the invention comprise FnIII scaffolds having BC loops which are randomized with the following consensus sequence: A-d-P-X-X-X-e-f-X-I-X-G, wherein X represents any amino acid, wherein (d) represents proline, glutamate or lysine, wherein (e) represents asparagine or glycine, and wherein (f) represents serine or glycine.

In one embodiment, the libraries of the invention comprise FnIII scaffolds having CD loops which are randomized with the following consensus sequence: X_(n), wherein X represents any amino acid, and wherein n=6, 7, 8, 9, or 10. In another embodiment, the scaffolds of the invention comprise an CD loop which is randomized with the following consensus sequence: X_(n), wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, and wherein n=7, 8, or 9.

In one embodiment the libraries of the invention comprise FnIII scaffolds having DE loops which are randomized with the following consensus sequence: X-X-X-X-X-X, wherein X represents any amino acid.

In one embodiment, the libraries of the invention comprise FnIII scaffolds having EF loops which are randomized with the following consensus sequence: X-b-L-X-P-X-c-X, wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, wherein (b) represents asparagine, lysine, arginine, aspartic acid, glutamic acid, or glycine, and wherein (c) represents isoleucine, threonine, serine, valine, alanine, or glycine.

In one embodiment, the libraries of the invention comprise FnIII scaffolds having FG loops which are randomized with the following consensus sequence: X-a-X-X-G-X-X-X-b, wherein X represents any amino acid, wherein (a) represents asparagine, threonine or lysine, and wherein (b) represents serine or alanine. In another embodiment, the libraries of the invention comprise FnIII scaffolds having FG loops which are randomized with the following consensus sequence: X-X-X-X-X-X-X-X-X (X₉), wherein X represents any amino acid.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the FnIII scaffolds comprise a Tn3 module. In another specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the FnIII scaffolds comprise a Tn3 module and wherein one or more of the beta strands of the Tn3 module comprise at least one amino acid substitution except that the cysteine in the C beta strand and the cysteine in the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be substituted.

In a specific embodiment, the libraries of the invention comprise FnIII scaffolds, wherein the scaffolds comprise the amino acid sequence: IEV(X_(AB))_(n)ALITW(X_(BC))_(n)CELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n)YEVSLIC(X_(FG))_(n)KET FTT, wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG) represent the amino acid residues present in the AB, BC, CD, DE, EF, and FG loops, respectively, wherein X₁ represents amino acid residue A or T, and wherein n=2-26 and m=1-9.

The invention further provides methods for identifying a recombinant FnIII scaffold that binds a target and has increased stability as compared to an FOI by screening the libraries of the invention, in particular the libraries comprising FnIII scaffolds wherein the FG loops are held to be at least one amino acid shorter than the cognate FG loop of the FOI.

In certain embodiments, the method for identifying a recombinant FnIII scaffold having increased protein stability as compared to an FOI, and which specifically binds a target, comprising:

-   -   a. contacting the target ligand with a library of the invention         under conditions suitable for forming a scaffold:target ligand         complex, wherein the libraries comprise FnIII scaffolds having         FG loops that are held to be at least one amino acid shorter         than the cognate FG loop of the POI;     -   b. obtaining from the complex, the scaffold that binds the         target ligand;     -   c. determining if the stability of the scaffold obtained in         step (b) is greater than that of the FOI.

In one embodiment, in step (a) the scaffold library of the invention is incubated with immobilized target. In one embodiment, in step (b) the scaffold:target ligand complex is washed to remove non-specific binders, and the tightest binders are eluted under very stringent conditions and subjected to PCR to recover the sequence information. Methods useful for the determination of stability in step (c) have been described supra. It is specifically contemplated that the binders and/or sequence information obtained in step (b) can be used to create a new library using the methods disclosed herein or known to one of skill in the art, which may be used to repeat the selection process, with or without further mutagenesis of the sequence. In some embodiments, a number of rounds of selection may be performed until binders of sufficient affinity for the antigen are obtained.

A further embodiment of the invention is a collection of isolated nucleic acid molecules encoding a library comprising the scaffolds of the invention and as described above.

Scaffolds of the invention may comprise codons encoded by the NHT codon scheme described in PCT Publication No: WO 2009/058379 or, alternatively, may comprise codons encoded by the NNK mixed codon scheme.

The scaffolds of the invention may be subjected to affinity maturation. In this art-accepted process, a specific binding protein is subject to a scheme that selects for increased affinity for a specific target (see Wu et al., Proc Natl Acad Sci USA. 95(11):6037-42). The resultant scaffolds of the invention may exhibit binding characteristics at least as high as compared to the scaffolds prior to affinity maturation.

The invention also provides methods of identifying the amino acid sequence of a protein scaffold capable of binding to target so as to form a scaffold:target complex. In one embodiment, the method comprises: a) contacting a library of the invention with an immobilized or separable target; b) separating the scaffold:target complexes from the free scaffolds; c) causing the replication of the separated scaffolds of (b) so as to result in a new polypeptide display library distinguished from that in (a) by having a lowered diversity and by being enriched in displayed scaffolds capable of binding the target; d) optionally repeating steps (a), and (b) with the new library of (c); and e) determining the nucleic acid sequence of the region encoding the displayed scaffold of a species from (d) and hence deducing the peptide sequence capable of binding to the target.

In another embodiment, the scaffolds of the invention may be further randomized after identification from a library screen. In one embodiment, methods of the invention comprise further randomizing at least one, at least two, at least three, at least four, at least five or at least six loops of a scaffold identified from a library using a method described herein. In another embodiment, the further randomized scaffold is subjected to a subsequent method of identifying a scaffold capable of binding a target. This method comprises (a) contacting said further randomized scaffold with an immobilized or separable target, (b) separating the further randomized scaffold:target complexes from the free scaffolds, (c) causing the replication of the separated scaffolds of (b), optionally repeating steps (a)-(c), and (d) determining the nucleic acid sequence of the region encoding said further randomized scaffold and hence, deducing the peptide sequence capable of binding to the target.

In a further embodiment, the further randomized scaffolds comprise at least one, at least two, at least three, at least four, at least five, or at least six randomized loops which were previously randomized in the first library. In an alternate further embodiment, the further randomized scaffolds comprise at least one, at least two, at least three, at least four, at least five, or at least six randomized loops which were not previously randomized in the first library.

The invention also provides a method for obtaining at least two FnIII scaffolds that bind to at least one or more targets. This method allows for the screening of agents that act cooperatively to elicit a particular response. It may be advantageous to use such a screen when an agonistic activity requiring the cooperation of more than one scaffold is required (for example, but not limited to, agonism of a receptor belonging to the TNF receptor family). This method allows for the screening of cooperative agents without the reformatting of the library to form multimeric complexes. In one embodiment, the method of the invention comprises contacting a target ligand with a library of the invention under conditions that allow a scaffold:target ligand complex to form, engaging said scaffolds with a crosslinking agent (defined as an agent that brings together, in close proximity, at least two identical or distinct scaffolds) wherein the crosslinking of the scaffolds elicits a detectable response and obtaining from the complex, said scaffolds that bind the target. In a further embodiment, the crosslinking agent is a scaffold specific antibody, or fragment thereof, an epitope tag specific antibody of a fragment thereof, a dimerization domain, such as Fc region, a coiled coil motif (for example, but not limited to, a leucine zipper), a chemical crosslinker, or another dimerization domain known in the art.

The invention also provides methods of detecting a compound utilizing the scaffolds of the invention. Based on the binding specificities of the scaffolds obtained by library screening, it is possible to use such scaffolds in assays to detect the specific target in a sample, such as for diagnostic methods. In one embodiment, the method of detecting a compound comprises contacting said compound in a sample with a scaffold of the invention, under conditions that allow a compound: scaffold complex to form and detecting said scaffold, thereby detecting said compound in a sample. In further embodiments, the scaffold is labeled (i.e., radiolabel, fluorescent, enzyme-linked or colorimetric label) to facilitate the detection of the compound.

The invention also provides methods of capturing a compound utilizing the scaffolds of the invention. Based on the binding specificities of the scaffolds obtained by library screening, it is possible to use such scaffolds in assays to capture the specific target in a sample, such as for purification methods. In one embodiment, the method of capturing a compound in a sample comprises contacting said compound in a sample with a scaffold of the invention under conditions that allow the formation of a compound:scaffold complex and removing said complex from the sample, thereby capturing said compound in said sample. In further embodiments, the scaffold is immobilized to facilitate the removing of the compound:scaffold complex.

In some embodiments, scaffolds isolated from libraries of the invention comprise at least one, at least two, at least four, at least five, at least six, or more randomized loops. In some embodiments, isolated scaffold loop sequences may be swapped from a donor scaffold to any loop in a receiver scaffold (for example, an FG loop sequence from a donor scaffold may be transferred to any loop in a receiver scaffold). In specific embodiments, an isolated loop sequences may be transferred to the cognate loop in the receiving scaffold (for example, an FG loop sequence from a donor scaffold may be transferred to a receiver scaffold in the FG loop position). In some embodiments, isolated loop sequences may be “mix and matched” randomly with various receiver scaffolds.

In other embodiments, isolated scaffolds sequences may be identified by the loop sequence. For example, a library is used to pan against a particular target and an collection of specific binders are isolated. The randomized loop sequences may be characterized as specific sequences independently of the scaffold background (i.e., the scaffold that binds target X wherein said scaffold comprises an FG loop sequence of SEQ ID NO:X). In alternative embodiments, where a scaffold exhibits two loop sequences that bind target X, the loop sequences may be characterized as binding target X in the absence of the scaffold sequence. In other words, it is contemplated that scaffolds isolated from a library that bind a particular target may be expressed as the variable loop sequences that bind that target independent of the scaffold backbone. This process would be analogous to the concept of CDRs in variable regions of antibodies.

Generation of Tandem Repeats

Linking of tandem constructs may be generated by ligation of oligonucleotides at restriction sites using restriction enzymes known in the art, including but not limited to type II and type IIS restriction enzymes. Type II restriction enzymes cut within their recognition sequence while type IIS restriction enzymes cut outside their recognition sequence to one side. In one embodiment for generating tandem repeats, type IIS enzymes are oriented so that cutting with them cleaves off their recognition site and leaves ends that can be joined together without generating recognition sites at the junction of two subunits. After ligation, both type II and type IIS sites remain at the ends. Additional subunits may be added by cutting with a type IIS restriction enzyme again and ligating. Alternatively, the clone may be cut with a type II restriction enzyme and ligated into a vector.

The multimeric scaffolds of the invention may comprise a linker at the C-terminus and/or the N-terminus and/or between domains as described herein. Further, scaffolds of the invention comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or polypeptide scaffolds may be fused or conjugated to a dimerization domain, including but not limited to an antibody moiety selected from:

-   -   (i) a Fab fragment, having VL, CL, VH and CH1 domains;     -   (ii) a Fab′ fragment, which is a Fab fragment having one or more         cysteine residues at the C-terminus of the CH1 domain;     -   (iii) a Fd′ fragment having VH and CH1 domains;     -   (iv) a Fd′ fragment having VH and CH1 domains and one or more         cysteine residues at the C-terminus of the CH1 domain;     -   (v) a Fv fragment having the VL and VH domains of a single arm         of an antibody;     -   (vi) a dAb fragment (Ward et al., Nature 341, 544-546 (1989))         which consists of a VH domain;     -   (vii) isolated CDR regions;     -   (viii) F(ab′)₂ fragments, a bivalent fragment including two Fab′         fragments linked by a disulphide bridge at the hinge region;     -   (ix) single chain antibody molecules (e.g., single chain Fv;         scFv) (Bird et al., Science 242:423-426 (1988); and Huston et         al., PNAS (USA) 85:5879-5883 (1988));     -   (x) a “diabody” with two antigen binding sites, comprising a         heavy chain variable domain (VH) connected to a light chain         variable domain (VL) in the same polypeptide chain (see, e.g.,         EP Patent Publication No. 404,097; WO93/11161; and Hollinger et         al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993));     -   (xi) a “linear antibody” comprising a pair of tandem Fd segments         (VH-CH1-VH-CH1) which, together with complementary light chain         polypeptides, form a pair of antigen binding regions (Zapata et         al. Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No.         5,641,870);     -   (xii) a full length antibody; and     -   (xiii) an Fc region comprising CH2-CH3, which may further         comprise all or a portion of a hinge region and/or a CH1 region.         Various valency, affinity, and spatial orientation schemes are         exemplified below in the Examples.

Scaffold Production

Recombinant expression of a scaffold of the invention requires construction of an expression vector containing a polynucleotide that encodes the scaffold. Once a polynucleotide encoding a scaffold has been obtained, the vector for the production of scaffold may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing a scaffold encoding nucleotide sequence are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing scaffold polypeptide coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding a scaffold of the invention, operably linked to a promoter.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce a scaffold of the invention. Thus, the invention includes host cells containing a polynucleotide encoding a scaffold of the invention, operably linked to a heterologous promoter. Suitable host cells include, but are not limited to, microorganisms such as bacteria (e.g., E. colit and B. subtilis).

A variety of host-expression vector systems may be utilized to express the scaffolds of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express a scaffold of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing scaffold coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing scaffold coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing scaffold coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing scaffold coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

Expression vectors containing inserts of a gene encoding a scaffold of the invention can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of “marker” gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a gene encoding a peptide, polypeptide, protein or a fusion protein in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted gene encoding the peptide, polypeptide, protein or the fusion protein, respectively. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain “marker” gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of a nucleotide sequence encoding an antibody or fusion protein in the vector. For example, if the nucleotide sequence encoding the scaffold is inserted within the marker gene sequence of the vector, recombinants containing the gene encoding the scaffold insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the gene product (e.g., scaffold or multimer thereof) expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the protein in in vitro assay systems, e.g., binding, agonistic or antagonistic properties of the scaffold.

Methods useful for the production of scaffolds of the invention are disclosed, for example, in Publication No: WO 2009/058379.

Scaffold Purification

Once a scaffold of the invention has been produced by recombinant expression, it may be purified by any method known in the art for purification of a protein, for example, by chromatography (e.g., metal-chelate chromatography, ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

The highly stable nature of the scaffolds of the invention allow for variations on purification schemes. For example, the thermal stability exhibited by the scaffolds of the invention allow for the heating of the crude lysate comprising the scaffolds to remove the bulk of the host cell proteins by denaturation. The high protease resistance exhibited by the scaffolds of the invention allows for the rapid degradation of host cell proteins in crude lysates prior to any purification steps. Also, the pH tolerance exhibited by some scaffolds of the invention allows for the selective precipitation of host cell proteins in the crude lysate by lowering or raising the pH prior to any purification steps. A combination of any of the above may be used in an effort to remove bulk host cell proteins from the crude lysate.

Production of the scaffolds of the invention in the research laboratory can be scaled up to produce scaffolds in analytical scale reactors or production scale reactors, as described in U.S. Patent Application Publ. No. US 2010/0298541 A1.

Scalable Production of Secreted Scaffolds

The scaffolds of the invention may be produced intracellularly or as a secreted form. In some embodiments, the secreted scaffolds are properly folded and fully functional. The production of secreted scaffolds comprises the use of a Ptac promoter and an oppA signal. The scaffold expressed in a prokaryotic host cell is secreted into the periplasmic space of the prokaryotic host cell into the media. Scaffolds of the invention may act as carrier molecules for the secretion of peptides and/or proteins into the cell culture media or periplasmic space of a prokaryotic cell.

In an effort to obtain large quantities, scaffolds of the invention may be produced by a scalable process (hereinafter referred to as “scalable process of the invention”). In some embodiments, scaffolds may be produced by a scalable process of the invention in the research laboratory that may be scaled up to produce the scaffolds of the invention in analytical scale bioreactors (for example, but not limited to 5 L, 10 L, 15 L, 30 L, or 50 L bioreactors). In other embodiments, the scaffolds may be produced by a scalable process of the invention in the research laboratory that may be scaled up to produce the scaffolds of the invention in production scale bioreactors (for example, but not limited to 75 L, 100 L, 150 L, 300 L, or 500 L). In some embodiments, the scalable process of the invention results in little or no reduction in production efficiency as compared to the production process performed in the research laboratory.

In some embodiments, the scalable process of the invention produces multimeric scaffolds at production efficiency of about 10 mg/L, about 20 m/L, about 30 mg/L, about 50 mg/L, about 75 mg/L, about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L, about 200 mg/L, about 250 mg/L, about 300 mg/L or higher.

In other embodiments, the scalable process of the invention produces multimeric scaffolds at a production efficiency of at least about 10 mg/L, at least about 20 m/L, at least about 30 mg/L, at least about 50 mg/L, at least about 75 mg/L, at least about 100 mg/L, at least about 125 mg/L, at least about 150 mg/L, at least about 175 mg/L, at least about 200 mg/L, at least about 250 mg/L, at least about 300 mg/L or higher.

In other embodiments, the scalable process of the invention produces multimeric scaffolds at a production efficiency from about 10 mg/L to about 300 mg/L, from about 10 mg/L to about 250 mg/L, from about 10 mg/L to about 200 mg/L, from about 10 mg/L to about 175 mg/L, from about 10 mg/L to about 150 mg/L, from about 10 mg/L to about 100 mg/L, from about 20 mg/L to about 300 mg/L, from about 20 mg/L to about 250 mg/L, from about 20 mg/L to about 200 mg/L, from 20 mg/L to about 175 mg/L, from about 20 mg/L to about 150 mg/L, from about 20 mg/L to about 125 mg/L, from about 20 mg/L to about 100 mg/L, from about 30 mg/L to about 300 mg/L, from about 30 mg/L to about 250 mg/L, from about 30 mg/L to about 200 mg/L, from about 30 mg/L to about 175 mg/L, from about 30 mg/L to about 150 mg/L, from about 30 mg/L to about 125 mg/L, from about 30 mg/L to about 100 mg/L, from about 50 mg/L to about 300 mg/L, from about 50 mg/L to about 250 mg/L, from about 50 mg/L to about 200 mg/L, from 50 mg/L to about 175 mg/L, from about 50 mg/L to about 150 mg/L, from about 50 mg/L to about 125 mg/L, or from about 50 mg/L to about 100 mg/L.

In some embodiments, the scalable process of the invention produces scaffolds at production efficiency of about 1 g/L, about 2 g/L, about 3 g/L, about 5 g/L, about 7.5 g/L, about 10 g/L, about 12.5 g/L, about 15.0 g/L, about 17.5 g/L, about 20 g/L, about 25 g/L, about 30 g/L, or higher.

In other embodiments, the scalable process of the invention produces scaffolds at a production efficiency of at least about 1 g/L, at least about 2 g/L, at least about 3 g/L, at least about 5 g/L, at least about 7.5 g/L, at least about 10 g/L, at least about 12.5 g/L, at least about 15 g/L, at least about 17.5 g/L, at least about 20 g/L, at least about 25 g/L, at least about 30 g/L, or higher.

Linkers

The scaffolds of the invention are linked by protein and/or nonprotein linkers, wherein each linker is fused to at least two scaffolds of the invention. Choosing a suitable linker for a specific case where two or more scaffolds of the invention are to be connected depends on a variety of parameters including, e.g., the nature of the FnIII monomer domains, the stability of the peptide linker towards proteolysis and oxidation, conformational constrains to guide multimer folding, and/or conformational constraints related to the desired biological activity of the scaffold.

A suitable linker can consist of a protein linker, a nonprotein linker, and combinations thereof. Combinations of linkers can be homomeric or heteromeric. In some embodiments, a multimeric FnIII scaffold of the invention comprises a plurality of FnIII scaffolds of the invention wherein are all the linkers are identical. In other embodiments, a multimeric FnIII scaffold of the invention comprises a plurality of FnIII scaffolds of the invention wherein at least one of the linkers is functionally or structurally different from the rest of the linkers. In some embodiments, linkers can themselves contribute to the activity of a multimeric FnIII scaffold by participating directly in the binding to a target.

In some embodiments, the protein linker is a polypeptide. In some embodiments, a linker polypeptide predominantly includes amino acid residues selected from the group consisting of Gly, Ser, Ala and Thr. For example, in some embodiments the peptide linker contains at least 75% (calculated on the basis of the total number of amino acid residues present in the peptide linker), at least 80%, at least 85% or at least 90% of amino acid residues selected from the group consisting of Gly, Ser, Ala and Thr. In some embodiments, the peptide linker consists of Gly, Ser, Ala and/or Thr residues only.

The linker polypeptide should have a length, which is adequate to link two or more monomer scaffolds of the invention or two or more multimeric scaffolds of the invention in such a way that they assume the correct conformation relative to one another so that they retain the desired activity.

In one embodiment, the polypeptide linker comprises 1 to about 1000 amino acids residues, 1 to about 50 amino acid residues, 1-25 amino acid residues, 1-20 amino acid residues, 1-15 amino acid residues, 1-10 amino acid residues, 1-5 amino acid residues, 1-3 amino acid residues. The invention further provides nucleic acids, such as DNA, RNA, or combinations of both, encoding the polypeptide linker sequence. The amino acid residues selected for inclusion in the polypeptide linker should exhibit properties that do not interfere significantly with the activity or function of the multimeric scaffold of the invention. Thus, a polypeptide linker should on the whole not exhibit a charge which would be inconsistent with the activity or function of the multimeric scaffold of the invention, or interfere with internal folding, or form bonds or other interactions with amino acid residues in one or more of the FnIII monomer domains which would seriously impede the binding of the multimeric scaffold of the invention to specific targets.

In some embodiments, randomization is used to obtain linkers that afford maximum stability and/or activity of a multimeric scaffold. In this process, conformationally flexible linkers are first used to find suitable combination of scaffolds of the invention, and the resulting multimeric scaffold is subsequently optimized by randomizing the amino acids residues in the polypeptide linkers.

The use of naturally occurring as well as artificial peptide linkers to connect polypeptides into novel linked fusion polypeptides is well known in the literature (Hallewell et al. (1989), J. Biol. Chem. 264, 5260-5268; Alfthan et al. (1995), Protein Eng 8, 725-731; Robinson & Sauer (1996), Biochemistry 35, 109-116; Khandekar et al. (1997), J. Biol. Chem. 272, 32190-32197; Fares et al. (1998), Endocrinology 139, 2459-2464; Smallshaw et al. (1999), Protein Eng. 12, 623-630; U.S. Pat. No. 5,856,456).

Accordingly, the linkers fusing two or more scaffolds of the invention are natural linkers (see, e.g., George & Hering a, Protein Eng. 11:871-879, 2002), artificial linkers, or combinations thereof. In some embodiments, the amino acid sequences of all peptide linkers present in a multimeric scaffold of the invention are identical. In other embodiments, the amino acid sequences of at least two of the peptide linkers present in a multimeric scaffold of the invention are different.

In some embodiments, a polypeptide linker possesses conformational flexibility. In some embodiments, a polypeptide linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues or 8-12 glycine residues. In some embodiments, a polypeptide linker comprises at least 50% glycine residues, at least 75% glycine residues, at least 80% glycine residues, or at least 85% glycine residues. In some embodiments, a polypeptide linker sequence comprises glycine residues only. In a specific embodiment, a polypeptide linker sequence comprises a (G-G-G-G-S)_(x) amino acid sequence where x is a positive integer. In another specific embodiment, a polypeptide linker sequence comprises a (G-A)_(x) sequence where x is a positive integer. In another specific embodiment, a polypeptide linker sequence comprises a (G-G-G-T-P-T)_(x) sequence where x is a positive integer. In still another specific embodiment, a polypeptide linker sequence comprises a (G-G-G-G-S-G-T-G-S-A-M-A-S)_(x) sequence where x is a positive integer.

In some embodiments, a polypeptide linker is an inherently unstructured natural or artificial polypeptide (see, e.g., Schellenberger et al., Nature Biotechnol. 27:1186-1190, 2009; see also, Sickmeier et al., Nucleic Acids Res. 35:D786-93, 2007).

In some embodiments, the conformational flexibility of a polypeptide linker is restricted by including one or more proline amino acid residues in the amino acid sequence of the polypeptide linker. Thus, in another embodiment of the invention, the polypeptide linker may comprise at least one proline residue in the amino acid sequence of the polypeptide linker. For example, the polypeptide linker has an amino acid sequence, wherein at least 25%, at least 50%, at least 75%, of the amino acid residues are proline residues. In one particular embodiment of the invention, the polypeptide linker comprises proline residues only.

In some embodiments, alpha-helix-forming linkers can be used, e.g., the Ala-(Glu-Ala-Ala-Ala-Lys)n-Ala linear linker (n=2-5) (see, e.g., Arai et al., Protein Eng 14:529-532, 2001) or alpha-helix-bundle linkers (see, e.g., Maeda et al., Anal. Biochem. 249:147-152, 1997). In other embodiments, Ser-rich linkers can be used, e.g., (Ser-4-Gly)_(n) (n>1) or (X4-Gly)_(n) (wherein up to two X's are Thr, the remaining X's are Ser, and n>1) (see U.S. Pat. No. 5,525,491). In other embodiments, (Gly-Ser)_(n), (Gly-Gly-Ser-Gly)_(n), or Gly-Ser-Ala-Thr linkers are used.

The peptide linker can be modified in such a way that an amino acid residue comprising an attachment group for a non-polypeptide moiety is introduced. Examples of such amino acid residues may be a cysteine residue (to which the non-polypeptide moiety is then subsequently attached) or the amino acid sequence may include an in vivo N-glycosylation site (thereby attaching a sugar moiety (in vivo) to the peptide linker). An additional option is to genetically incorporate non-natural amino acids using evolved tRNAs and tRNA synthetases (see, e.g., U.S. Patent Appl. Publ. No. 2003/0082575) into the monomer domains or linkers. For example, insertion of keto-tyrosine allows for site-specific coupling to expressed monomer domains or multimers.

In some embodiments, the amino acid sequences of all peptide linkers present in the polypeptide multimer are identical. Alternatively, the amino acid sequences of all peptide linkers present in the polypeptide multimer may be different.

Labeling or Conjugation of Scaffolds

The scaffolds of the invention can be used in non-conjugated form or conjugated to at least one of a variety of heterologous moieties to facilitate target detection or for imaging or therapy. The scaffolds of the can be labeled or conjugated either before or after purification, when purification is performed.

Many heterologous moieties lack suitable functional groups to which scaffolds of the invention can be linked. Thus, in some embodiments, the effector molecule is attached to the scaffold through a linker, wherein the linker contains reactive groups for conjugation. In some embodiments, the heterologous moiety conjugated to a scaffold of the invention can function as a linker. In other embodiments, the moiety is conjugated to the scaffold via a linker that can be cleavable or non-cleavable. In one embodiment, the cleavable linking molecule is a redox cleavable linking molecule, such that the linking molecule is cleavable in environments with a lower redox potential, such as the cytoplasm and other regions with higher concentrations of molecules with free sulfhydryl groups. Examples of linking molecules that may be cleaved due to a change in redox potential include those containing disulfides.

In some embodiments, scaffolds of the invention are engineered to provide reactive groups for conjugation. In such scaffolds, the N-terminus and/or C-terminus can also serve to provide reactive groups for conjugation. In other embodiments, the N-terminus can be conjugated to one moiety (such as, but not limited to PEG) while the C-terminus is conjugated to another moiety (such as, but not limited to biotin), or vice versa.

The term “polyethylene glycol” or “PEG” means a polyethylene glycol compound or a derivative thereof, with or without coupling agents, coupling or activating moieties (e.g., with thiol, triflate, tresylate, aziridine, oxirane, N-hydroxysuccinimide or a maleimide moiety). The term “PEG” is intended to indicate polyethylene glycol of a molecular weight between 500 and 150,000 Da, including analogues thereof, wherein for instance the terminal OH-group has been replaced by a methoxy group (referred to as mPEG).

The scaffolds of the invention can be derivatized with polyethylene glycol (PEG). PEG is a linear, water-soluble polymer of ethylene oxide repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights which typically range from about 500 daltons to about 40,000 daltons. In a specific embodiment, the PEGs employed have molecular weights ranging from 5,000 daltons to about 20,000 daltons. PEGs coupled to the scaffolds of the invention can be either branched or unbranched. (See, for example, Monfardini, C. et al. 1995 Bioconjugate Chem 6:62-69). PEGs are commercially available from Nektar Inc., Sigma Chemical Co. and other companies. Such PEGs include, but are not limited to, monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

Briefly, the hydrophilic polymer which is employed, for example, PEG, is capped at one end by an unreactive group such as a methoxy or ethoxy group. Thereafter, the polymer is activated at the other end by reaction with a suitable activating agent, such as cyanuric halides (for example, cyanuric chloride, bromide or fluoride), carbonyldiimidazole, an anhydride reagent (for example, a dihalo succinic anhydride, such as dibromosuccinic anhydride), acyl azide, p-diazoniumbenzyl ether, 3-(p-diazoniumphenoxy)-2-hydroxypropylether) and the like. The activated polymer is then reacted with a polypeptide as described herein to produce a polypeptide derivatized with a polymer. Alternatively, a functional group in the scaffolds of the invention can be activated for reaction with the polymer, or the two groups can be joined in a concerted coupling reaction using known coupling methods. It will be readily appreciated that the polypeptides of the invention can be derivatized with PEG using a myriad of other reaction schemes known to and used by those of skill in the art.

In other embodiments, scaffolds of the invention, analogs or derivatives thereof may be conjugated to a diagnostic or detectable agent. Such scaffolds can be useful for monitoring or prognosing the development or progression of a disease as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. Such diagnosis and detection can be accomplished by coupling the scaffold to detectable substances including, but not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to streptavidin/biotin and avidin/biotin; fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as but not limited to iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In,), and technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), samarium (¹⁵³Sm), lutetium (¹⁷⁷Lu), gadolinium (¹⁵⁹Gd, ¹⁵³Gd), promethium (¹⁴⁹Pm), lanthanum (¹⁴⁰La), ytterbium (¹⁷⁵Yb, ¹⁶⁹Yb), holmium (¹⁶⁶Ho), yytrium (⁹⁰Y), scandium (⁴⁷Sc), rhenium (¹⁸⁶Re, ¹⁸⁸Re), praseodymium (¹⁴²Pr), rhodium (¹⁰⁵Rh) ruthenium (⁹⁷Ru), germanium (⁶⁸Ge), cobalt (⁵⁷Co), zinc (⁶⁵Zn), strontium (⁸⁵Sr), phosphorus (³²P), chromium (⁵¹Cr), manganese (⁵⁴Mn), selenium (⁷⁵Se), tin (¹¹³Sn), and indium (¹¹⁷In); positron emitting metals using various positron emission topographies, nonradioactive paramagnetic metal ions, and molecules that are radiolabeled or conjugated to specific radioisotopes.

The present invention further encompasses uses of scaffolds conjugated to a therapeutic moiety. A scaffold may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Therapeutic moieties include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustihe (BCNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), Auristatin molecules (e.g., auristatin PHE, bryostatin 1, and solastatin 10; see Woyke et al., Antimicrob. Agents Chemother. 46:3802-8 (2002), Woyke et al., Antimicrob. Agents Chemother. 45:3580-4 (2001), Mohammad et al., Anticancer Drugs 12:735-40 (2001), Wall et al., Biochem. Biophys. Res. Commun. 266:76-80 (1999), Mohammad et al., Int. J. Oncol. 15:367-72 (1999), all of which are incorporated herein by reference), hormones (e.g., glucocorticoids, progestins, androgens, and estrogens), DNA-repair enzyme inhibitors (e.g., etoposide or topotecan), kinase inhibitors (e.g., compound ST1571, imatinib mesylate (Kantarjian et al., Clin Cancer Res. 8(7):2167-76 (2002)), cytotoxic agents (e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof) and those compounds disclosed in U.S. Pat. Nos. 6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196, 6,218,410, 6,218,372, 6,057,300 6,034,053, 5,985,877, 5,958,769, 5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, 5,587,459), farnesyl transferase inhibitors (e.g., R1 15777, BMS-214662 and those disclosed by, for example, U.S. Pat. Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905, 6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338, 6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786, 6,169,096, 6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465, 6,124,295, 6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853, 6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574, and 6,040,305), topoisomerase inhibitors (e.g., camptothecin; irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI 147211); DX-8951f; IST-622; rubitecan; pyrazoloacridine; XR-5000; saintopin; UCE6; UCE1022; TAN-1518A; TAN-1518B; KT6006; KT6528; ED-110; NB-506; ED-110; NB-506; and rebeccamycin); bulgarein; DNA minor groove binders such as Hoescht dye 33342 and Hoechst dye 33258; nitidine; fagaronine; epiberberine; coralyne; beta-lapachone; BC-4-1; and pharmaceutically acceptable salts, solvates, clathrates, and prodrugs thereof. See, e.g., Rothenberg, M. L., Annals of Oncology 8:837-855 (1997); and Moreau, P., et al., J. Med. Chem. 41:1631-1640 (1998); bisphosphonates (e.g., alendronate, cimadronte, clodronate, tiludronate, etidronate, ibandronate, neridronate, olpandronate, risedronate, piridronate, pamidronate, zolendronate) HMG-CoA reductase inhibitors, statins (e.g., lovastatin, simvastatin, atorvastatin (Lipitor™), pravastatin, fluvastatin (Lescol™, cerivastatin, and rosuvastatin)), antisense oligonucleotides (e.g., those disclosed in the U.S. Pat. Nos. 6,277,832, 5,998,596, 5,885,834, 5,734,033, and 5,618,709), immunomodulators (e.g., antibodies and cytokines), and adenosine deaminase inhibitors (e.g., fludarabine phosphate and 2-chlorodeoxyadenosine).

Further, a scaffold may be conjugated to a therapeutic moiety or drug moiety that modifies a given biological response. Therapeutic moieties or drug moieties are not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzyme, an antibody, a toxin (e.g., abrin, ricin A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as a tumor necrosis factor (e.g., TNF-alpha, TNF-beta), an interferon (e.g., α-interferon, β-interferon), a nerve growth factor, a platelet derived growth factor, a tissue plasminogen activator, an apoptotic agent (e.g., TNF-alpha, TNF-beta, AIM I (see, International publication No. WO 97/33899), AIM II (see, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., 1994, J. Immunol., 6:1567-1574), and VEGI (see, International publication No. WO 99/23105)), a thrombotic agent or an anti-angiogenic agent (e.g., angiostatin, endostatin or a component of the coagulation pathway (e.g., tissue factor)); or, a biological response modifier such as, for example, a lymphokine (e.g., interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”)), a growth factor (e.g., growth hormone (“GH”)), or a coagulation agent (e.g., calcium, vitamin K, tissue factors, such as but not limited to, Hageman factor (factor XII), high-molecular-weight kininogen (HMWK), prekallikrein (PK), coagulation proteins-factors II (prothrombin), factor V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid, fibrinopeptides A and B from the α and β chains of fibrinogen, fibrin monomer).

Moreover, a scaffold can be conjugated to therapeutic moieties such as a radioactive metal ion, such as alpha-emitters such as ²¹³Bi or macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, ¹³¹In, ¹³¹Lu, ¹³¹Y, ¹³¹Ho, ¹³¹Sm, to polypeptides. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″″-tetraacetic acid (DOTA) which can be attached to the scaffold via a linker molecule. Such linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50, each incorporated by reference in their entireties.

Techniques for conjugating therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56. (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol. Rev. 62: 119-58. Similar approaches may be adapted for use with scaffolds of the invention.

The therapeutic moiety or drug conjugated to a scaffold of the invention should be chosen to achieve the desired prophylactic or therapeutic effect(s) for a particular disorder in a subject. A clinician or other medical personnel should consider the following when deciding on which therapeutic moiety or drug to conjugate to a scaffold: the nature of the disease, the severity of the disease, and the condition of the subject.

Assaying Scaffolds

The scaffolds of the invention may be assayed for specific binding to a target by any method known in the art. Representative assays which can be used, include but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, to name but a few. Such assays are routine and known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).

The binding affinity and other binding properties of a scaffold to an antigen may be determined by a variety of in vitro assay methods known in the art including for example, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA; or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE® analysis), and other methods such as indirect binding assays, competitive binding assays, gel electrophoresis and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999).

In some embodiments, scaffolds of the invention specifically bind a target with specific kinetics. In some embodiments, scaffolds of the invention may have a dissociation constant or K_(d) (k_(off)/k_(on)) of less than 1×10⁻² M, 1×10^(−M), 1×10⁻⁴M, 1×10⁻⁵M, 1×10⁻⁶M, 1×10⁻⁷M, 1×10⁻⁸M, 1×10⁻⁹M, 1×10⁻¹⁰M, 1×10⁻¹¹M, 1×10⁻¹²M, 1×10⁻¹³M, 1×10⁻¹⁴M or less than 1×10⁻¹⁵M. In specific embodiments, scaffolds of the invention have a K_(d) of 500 μM, 100 μM, 100 μM, 500 nM, 100 nM, 1 nM, 500 pM, 100 pM or less as determined by a BIAcore Assay® or by other assays known in the art. In an alternative embodiment, the affinity of the scaffolds of the invention is described in terms of the association constant (K_(a)), which is calculated as the ratio k_(on)/k_(off), of at least 1×10²M⁻¹, 1×10³M⁻¹, 1×10⁴M⁻¹, 1×10⁵M⁻¹, 1×10⁶M⁻¹, 1×10⁷M⁻¹, 1×10⁸M⁻¹, 1×10⁹M⁻¹, 1×10¹⁰M⁻¹ 1×10¹¹M⁻¹ 1×10¹²M⁻¹, 1×10¹³M⁻¹, 1×10¹⁴M⁻¹, 1×10¹⁵M⁻¹, or at least 5×10¹⁵ M⁻¹.

In certain embodiments the rate at which the scaffolds of the invention dissociate from a target epitope may be more relevant than the value of the K_(d) or the K_(a). In some embodiments, the scaffolds of the invention have a k_(off) of less than 10⁻³ s⁻¹, less than 5×10⁻³ s⁻¹, less than 10⁻⁴ s⁻¹, less than 5×10⁻⁴ s⁻¹, less than 10⁻⁵ s⁻¹, less than 5×10⁻⁵ s⁻¹, less than 10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁷ s⁻¹, less than 5×10⁻⁷ s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁹ s⁻¹, less than 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹.

In certain other embodiments, the rate at which the scaffolds of the invention associate with a target epitope may be more relevant than the value of the K_(d) or the K_(a). In this instance, the scaffolds of the invention bind to a target with a k_(on) rate of at least 10⁵ M⁻¹s⁻¹, at least 5×10⁵M⁻¹s⁻¹, at least 10⁶ M⁻¹ s⁻¹, at least 5×10⁶ M⁻¹s⁻¹, at least 10⁷ M⁻¹s⁻¹, at least 5×10⁷M⁻¹s⁻¹, or at least 10⁸M⁻¹s⁻¹, or at least 10⁹ M⁻¹s⁻¹.

Scaffolds of the invention may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Assays for Detecting Soluble, Secreted Polypeptides

In a specific embodiment, the invention provides an improved ELISA method for detecting soluble recombinant polypeptides secreted in culture media. In some embodiments, the recombinant polypeptide is a recombinant Fn type III variant. In one embodiment, the method for detecting a soluble recombinant fibronectin type III variant comprises:

-   -   (a) reacting culture media containing an expressed polypeptide         with an immobilized antibody which binds to the polypeptide,     -   (b) reacting the polypeptide with a target conjugated to an         enzyme under conditions suitable for binding,     -   (c) reacting the bound conjugate target to a substrate wherein a         signal is generated, and     -   (d) measuring the signal intensity.

In another embodiment, the method comprises:

-   -   (a) reacting culture media containing an expressed variant with         an antibody which binds the variant, wherein the antibody is         immobilized to a solid support;     -   (b) washing the immobilized support with buffer solution;     -   (c) reacting the variant with a target conjugated to a first         member of a binding pair;     -   (d) washing the immobilized support with buffer solution;     -   (e) reacting said first member with a second member of a binding         pair, wherein said second member is conjugated to an enzyme;     -   (f) reacting said enzyme with a substrate, wherein a signal is         generated; and     -   (g) measuring the intensity of said signal, wherein signal         intensity correlates with binding affinity. In one embodiment,         the method comprises detecting a secreted polypeptide or         secreted variant in crude culture media.

In a specific embodiment, the method comprises detecting a secreted polypeptide or secreted variant in crude culture media. In a specific embodiment, the signal intensity varies by less than 40%, less than 30%, less than 20%, or less than 19%.

In one embodiment, the ELISA method is performed in a high throughput or ultrahigh throughput format using assay plates of at least 96 wells. In a specific embodiment, a 384 well assay plate or a 1536 well assay plate is used.

In another embodiment, the method detects a variant comprising a heterologous amino acid sequence, including but not limited to: a poly(his) tag, a hemagglutinin (HA) tag, a FLAG tag, a Strep-tag, a myc tag, or a V5 tag.

In yet another embodiment, the first member of the binding pair is biotin and the second member of the binding pair is streptavidin or avidin.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, for example, but not limited to, a pharmaceutical composition, containing one or a combination of scaffolds or multimeric scaffolds of the present invention, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of, for example, but not limited to two or more different scaffolds of the invention. For example, a pharmaceutical composition of the invention may comprise a combination of scaffolds that bind to different epitopes on the target antigen or that have complementary activities. In a specific embodiment, a pharmaceutical composition comprises a multimeric scaffold of the invention.

Pharmaceutical compositions of the invention also can be administered in combination therapy, such as, combined with other agents. For example, the combination therapy can include a scaffold of the present invention combined with at least one other therapy wherein the therapy may be immunotherapy, chemotherapy, radiation treatment, or drug therapy.

The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be suitable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In one embodiment the compositions (e.g., liquid formulations) of the invention are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined inside a microorganism and are released when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxins from intravenously administered pharmaceutical drug solutions. The Food & Drug Administration (“FDA”) has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one hour period for intravenous drug applications (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins are administered in amounts of several hundred or thousand milligrams per kilogram body weight it is advantageous to remove even trace amounts of endotoxin. In one embodiment, endotoxin and pyrogen levels in the composition are less than 10 EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0.01 EU/mg, or less than 0.001 EU/mg. In another embodiment, endotoxin and pyrogen levels in the composition are less than about 10 EU/mg, or less than about 5 EU/mg, or less than about 1 EU/mg, or less than about 0.1 EU/mg, or less than about 0.01 EU/mg, or less than about 0.001 EU/mg.

Pharmaceutical Dosing and Administration

To prepare pharmaceutical or sterile compositions including a scaffold of the invention, a scaffold is mixed with a pharmaceutically acceptable carrier or excipient. Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

Selecting an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. In certain embodiments, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003) New Engl J. Med. 348:601-608; Milgrom, et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon, et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz, et al. (2000) New Engl. J. Med. 342:613-619; Ghosh, et al. (2003) New Engl. J. Med. 348:24-32; Lipsky, et al. (2000) New Engl. J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

Scaffolds of the invention can be provided by continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1-7 times per week. Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, or by inhalation. A specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects. A total weekly dose may be at least 0.05 μg/kg body weight, at least 0.2 μg/kg, at least 0.5 μg/kg, at least 1 μg/kg, at least 10 μg/kg, at least 100 μg/kg, at least 0.2 mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg, at least 10 mg/kg, at least 25 mg/kg, or at least 50 mg/kg (see, e.g., Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al. (20003) Cancer Immunol. Immunother. 52:133-144). The desired dose of a small molecule therapeutic, e.g., a peptide mimetic, protein scaffold, natural product, or organic chemical, is about the same as for an antibody or polypeptide, on a moles/kg body weight basis.

The desired plasma concentration of a small molecule or scaffold therapeutic is about the same as for an antibody, on a moles/kg body weight basis. The dose may be at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, or at least 100 μg. The doses administered to a subject may number at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or more.

For scaffolds of the invention, the dosage administered to a patient may be 0.0001 mg/kg to 100 mg/kg of the patient's body weight. The dosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight.

The dosage of the scaffolds of the invention may be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg/kg. The dosage of the scaffolds of the invention may be 150 μg/kg or less, 125 μg/kg or less, 100 μg/kg or less, 95 μg/kg or less, 90 μg/kg or less, 85 μg/kg or less, 80 μg/kg or less, 75 μg/kg or less, 70 μg/kg or less, 65 μg/kg or less, 60 μg/kg or less, 55 μg/kg or less, 50 μg/kg or less, 45 μg/kg or less, 40 μg/kg or less, 35 μg/kg or less, 30 μg/kg or less, 25 μg/kg or less, 20 μg/kg or less, 15 μg/kg or less, 10 μg/kg or less, 5 μg/kg or less, 2.5 μg/kg or less, 2 μg/kg or less, 1.5 μg/kg or less, 1 μg/kg or less, 0.5 μg/kg or less, or 0.5 μg/kg or less of a patient's body weight.

Unit dose of the scaffolds of the invention may be 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

The dosage of the scaffolds of the invention may achieve a serum titer of at least 0.1 μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, at least 200 μg/ml, at least 225 μg/ml, at least 250 μg/ml, at least 275 μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375 μg/ml, or at least 400 μg/ml in a subject. Alternatively, the dosage of the scaffolds of the invention may achieve a serum titer of at least 0.1 μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least, 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, at least 200 μg/ml, at least 225 μg/ml, at least 250 μg/ml, at least 275 μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375 μg/ml, or at least 400 μg/ml in the subject.

Doses of scaffolds of the invention may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects (see, e.g., Maynard, et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

A composition of the present invention may also be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Selected routes of administration for scaffolds of the invention include without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracerebral, intraocular, intraocular, intraarterial, intracerebrospinal, intralesional intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, or by sustained release systems or an implant (see, e.g., Sidman et al. (1983) Biopolymers 22:547-556; Langer, et al. (1981) J. Biomed. Mater. Res. 15:167-277; Langer (1982) Chem. Tech. 12:98-105; Epstein, et al. (1985) Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang, et al. (1980) Proc. Natl. Acad. Sci. USA 77:4030-4034; U.S. Pat. Nos. 6,350,466 and 6,316,024). Alternatively, a composition of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference their entirety. In one embodiment, an antibody, combination therapy, or a composition of the invention is administered using Alkermes AIR™ pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).

If the scaffolds of the invention are administered in a controlled release or sustained release system, a pump may be used to achieve controlled or sustained release (see Langer, Chem. Tech. 12:98-105, 1982; Seflon, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al, 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:51 A). Polymeric materials can be used to achieve controlled or sustained release of the therapies of the invention (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol Sd. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253.

Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. A controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more scaffolds of the invention. See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology 39: 179-189, Song et al, 1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Pro. Int'l. Symp. Control. ReI. Bioact. Mater. 24:853-854, and Lam et al, 1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in their entirety.

The scaffolds of the invention can be formulated for topical administration in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity, in some instances, greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in some instances, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.

If the scaffolds of the invention are administered intranasally, it can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Methods for co-administration or treatment with a second therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation, are well known in the art (see, e.g., Hardman, et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10^(th) ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.).

An effective amount of therapeutic may decrease the symptoms by at least 10%; by at least 20%; at least about 30%; at least 40%, or at least 50%.

Additional therapies (e.g., prophylactic or therapeutic agents), which can be administered in combination with the scaffolds of the invention may be administered to a subject concurrently. The term “concurrently” is not limited to the administration of therapies (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather it is meant that a pharmaceutical composition comprising scaffolds of the invention are administered to a subject in a sequence and within a time interval such that the scaffolds of the invention can act together with the other therapy or therapies to provide an increased benefit than if they were administered otherwise. For example, each therapy may be administered to a subject at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. Each therapy can be administered to a subject separately, in any appropriate form and by any suitable route. In various embodiments, the therapies (e.g., prophylactic or therapeutic agents) are administered to a subject less than 15 minutes, less than 30 minutes, less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart. The two or more therapies may be administered within one same patient visit.

The scaffolds of the invention and the other therapies may be cyclically administered. Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, optionally, followed by the administration of a third therapy (e.g., prophylactic or therapeutic agent) for a period of time and so forth, and repeating this sequential administration, i.e., the cycle in order to reduce the development of resistance to one of the therapies, to avoid or reduce the side effects of one of the therapies, and/or to improve the efficacy of the therapies.

In certain embodiments, the scaffolds of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al); mannosides (Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); pI20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J J. Killion; I J. Fidler (1994; Immunomethods 4:273.

The prophylactic or therapeutic agents of the combination therapies can be administered to a subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies can be administered concurrently to a subject in separate pharmaceutical compositions. The prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.

Methods of Using Scaffolds

The scaffolds of the present invention have in vitro and in vivo diagnostic and therapeutic utilities. For example, these molecules can be administered to cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a variety of disorders.

The invention also provides methods of using the scaffolds of the invention. The present invention also encompasses the use of the scaffolds of the invention for the prevention, diagnosis, management, treatment or amelioration of one or more symptoms associated with diseases, disorders of diseases or disorders, including but not limited to cancer, inflammatory and autoimmune diseases, infectious diseases either alone or in combination with other therapies. The invention also encompasses the use of the scaffolds of the invention conjugated or fused to a moiety (e.g., therapeutic agent or drug) for prevention, management, treatment or amelioration of one or more symptoms associated with diseases, disorders or infections, including but not limited to cancer, inflammatory and autoimmune diseases, infectious diseases either alone or in combination with other therapies.

Also, many cell surface receptors activate or deactivate as a consequence of cross-linking of sub units. The proteins of the invention may be used to stimulate or inhibit a response in a target cell by cross-linking of cell surface receptors. In another embodiment, the scaffolds of the invention of the invention may be used to block the interaction of multiple cell surface receptors with antigens. In another embodiment, the scaffolds of the invention may be used to strengthen the interaction of multiple cell surface receptors with antigens. In another embodiment, it may be possible to crosslink homo- or heterodimers of a cell surface receptor using the scaffolds of the invention containing binding domains that share specificity for the same antigen, or bind two different antigens. In another embodiment, the proteins of the invention could be used to deliver a ligand, or ligand analogue to a specific cell surface receptor.

The invention also provides methods of targeting epitopes not easily accomplished with traditional antibodies. For example, in one embodiment, the scaffolds and of the invention may be used to first target an adjacent antigen and while binding, another binding domain may engage the cryptic antigen.

The invention also provides methods of using the scaffolds to bring together distinct cell types. In one embodiment, the proteins of the invention may bind a target cell with one binding domain and recruit another cell via another binding domain. In another embodiment, the first cell may be a cancer cell and the second cell is an immune effector cell such as an NK cell. In another embodiment, the scaffolds of the invention may be used to strengthen the interaction between two distinct cells, such as an antigen presenting cell and a T cell to possibly boost the immune response.

The invention also provides methods of using the scaffolds to ameliorate, treat, or prevent cancer or symptoms thereof. In one embodiment, methods of the invention are useful in the treatment of cancers of the head, neck, eye, mouth, throat, esophagus, chest, skin, bone, lung, colon, rectum, colorectal, stomach, spleen, kidney, skeletal muscle, subcutaneous tissue, metastatic melanoma, endometrial, prostate, breast, ovaries, testicles, thyroid, blood, lymph nodes, kidney, liver, pancreas, brain, or central nervous system.

The invention also provides methods of using the scaffolds to deplete a cell population. In one embodiment, methods of the invention are useful in the depletion of the following cell types: eosinophil, basophil, neutrophil, T cell, B cell, mast cell, monocytes and tumor cell.

The invention also provides methods of using scaffolds to inactivate, inhibit, or deplete cytokines. In one embodiment, methods of the invention are useful in the inactivation, inhibition, or depletion of at least one of the following cytokines: TNF-α, TGF-β, C5a, fMLP, Interferon alpha (including subtypes 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17 and 21), Interferon beta, Interferon omega, Interferon gamma, interleukins IL-1-33, CCL1-28, CXCL 1-17, and CX3CL1.

The invention also provides methods of using the scaffolds to inactivate various infections agents such as viruses, fungi, eukaryotic microbes, and bacteria. In some embodiments the scaffolds of the invention may be used to inactivate RSV, hMPV, PIV, or influenza viruses. In other embodiments, the scaffolds of the invention may be used to inactivate fungal pathogens, such as, but not limited to members of Naegleria, Aspergillus, Blastomyces, Histoplasma, Candida or Tinea genera. In other embodiments, the scaffolds of the invention may be used to inactivate eukaryotic microbes, such as, but not limited to members of Giardia, Toxoplasma, Plasmodium, Trypanosoma, and Entamoeba genera. In other embodiments, the scaffolds of the invention may be used to inactivate bacterial pathogens, such as but not limited to members of Staphylococcus, Streptococcus, Pseudomonas, Clostridium, Borrelia, Vibrio and Neisseria genera.

The invention also provides methods of using scaffolds proteins as diagnostic reagents. The proteins of the invention may be useful in kits or reagents where different antigens need to be efficiently captured concurrently.

The proteins of the invention and compositions comprising the same are useful for many purposes, for example, as therapeutics against a wide range of chronic and acute diseases and disorders including, but not limited to, cancer. Examples of cancers that can be prevented, managed, treated or ameliorated in accordance with the methods of the invention include, but are not limited to, cancer of the head, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, and brain. Additional cancers include, but are not limited to, the following: leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblasts, promyelocytic, myelomonocytic, monocytic, erythroleukemic leukemias and myclodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective tissue sarcomas such as but not limited to bone sarcoma, myeloma bone disease, multiple myeloma, cholesteatoma-induced bone osteosarcoma, Paget's disease of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease (including juvenile Paget's disease) and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polyploid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to papillary, nodular, and diffuse; lung cancers such as non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocyte, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers such as but not limited to renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/or ureter); Wilms' tumor; bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., inc., United States of America).

It is also contemplated that cancers caused by aberrations in apoptosis can also be treated by the methods and compositions of the invention. Such cancers may include, but not be limited to, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes.

The proteins of the invention and compositions comprising the same are useful for many purposes, for example, as therapeutics against a wide range of chronic and acute diseases and disorders including, but not limited to, autoimmune and/or inflammatory diseases. The compositions and methods of the invention described herein are useful for the prevention or treatment of autoimmune disorders and/or inflammatory disorders. Examples of autoimmune and/or inflammatory disorders include, but are not limited to, antiphospholipid syndrome, arthritis, atherosclerosis, anaphylactic shock, autoimmune Addison's disease, alopecia greata, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis, autoimmune orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic inflammation, Churg-Strauss syndrome, cicatrical pemphigoid, cold agglutinin disease, corneal and other tissue transplantation, CREST syndrome, Crohn's disease, cystic fibrosis, diabetic retinopathies, discoid lupus, endocarditis, endotoxic shock, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, hemangiomas, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura, IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, neovascular glaucoma, organ ischemia, pemphigus vulgaris, peritonitis, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, reperfusion injury, retrolental fibroplasia, rheumatoid arthritis, sarcoidosis, scleroderma, sepsis, septicemia, Sjogren's syndrome, spinal cord injury, stiff-man syndrome, systemic lupus erythematosus, takayasu arteritis, temporal arteritis/giant cell arteritis, thyroid hyperplasias, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.

Examples of inflammatory disorders include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacteria infections. The compositions and methods of the invention can be used with one or more conventional therapies that are used to prevent, manage or treat the above diseases.

The proteins of the invention and compositions comprising the same are useful for many purposes, for example, as therapeutics against a wide range of chronic and acute diseases and disorders including, but not limited to, infectious disease, including viral, bacterial and fungal diseases.

Examples of viral pathogens include but are not limited to: adenovirdiae (e.g., mastadenovirus and aviadeno virus), herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, and herpes simplex virus 6), leviviridae (e.g., levivirus, enterobacteria phase MS2, allolevirus), poxyiridae (e.g., chordopoxyirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporiipoxvirus, suipoxvirus, molluscipoxvirus, and entomopoxyirinae), papovaviridae (e.g., polyomavirus and papillomavirus), paramyxoviridae (e.g., paramyxovirus, parainfluenza virus 1, mobillivirus (e.g., measles virus), rubulavirus (e.g., mumps virus), pneumonovirinae (e.g., pneumovirus, human respiratory syncytial virus), and metapneumo virus (e.g., avian pneumovirus and human metapneumo virus)), picornaviridae (e.g., enterovirus, rhino virus, hepato virus (e.g., human hepatits A virus), cardiovirus, and apthovirus), reoviridae (e.g., orthoreovirus, orbivirus, rotavirus, cypovirus, fijivirus, phytoreo virus, and oryzavirus), retroviridae (e.g., mammalian type B retroviruses, mammalian type C retroviruses, avian type C retroviruses, type D retrovirus group, BLV-HTLV retroviruses, lentivirus (e.g. human immunodeficiency virus 1 and human immunodeficiency virus 2), spumavirus), flaviviridae (e.g., hepatitis C virus), hepadnaviridae (e.g., hepatitis B virus), togaviridae (e.g., alphavirus (e.g., sindbis virus) and rubivirus (e.g., rubella virus)), rhabdoviridae (e.g., vesiculovirus, lyssavirus, ephemerovirus, cytorhabdo virus, and necleorhabdo virus), arenaviridae (e.g., arenavirus, lymphocytic choriomeningitis virus, Ippy virus, and lassa virus), and coronaviridae (e.g., coronavirus and torovirus).

Examples of bacterial pathogens include but are not limited to: but not limited to, the Aquaspirillum family, Azospirillum family, Azotobacteraceae family, Bacteroidaceae family, Bartonella species, Bdellovibrio family, Campylobacter species, Chlamydia species (e.g., Chlamydia pneumoniae), Clostridium, Enterobacteriaceae family (e.g., Citrobacter species, Edwardsiella, Enterobacter aerogenes, Erwinia species, Escherichia coli, Hafnia species, Klebsiella species, Morganella species, Proteus vulgaris, Providencia, Salmonella species, Serratia marcescens, and Shigella flexneri), Gardinella family, Haemophilus influenzae, Halobacteriaceae family, Helicobacter family, Legionallaceae family, Listeria species, Methylococcaceae family, mycobacteria (e.g., Mycobacterium tuberculosis), Neisseriaceae family, Oceanospirillum family, Pasteurellaceae family, Pneumococcus species, Pseudomonas species, Rhizobiaceae family, Spirillum family, Spirosomaceae family, Staphylococcus (e.g., methicillin resistant Staphylococcus aureus and Staphylococcus pyrogenes), Streptococcus (e.g., Streptococcus enteritidis, Streptococcus fasciae, and Streptococcus pneumoniae), Vampirovibrio, Helicobacter family, and Vampirovibrio family.

Examples of fungal pathogens include, but are not limited to: Absidia species (e.g., Absidia corymbifera and Absidia ramosa), Aspergillus species, (e.g., Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus), Basidiobolus ranarum, Blastomyces dermatitidis, Candida species (e.g., Candida albicans, Candida glabrata, Candida kerr, Candida krusei, Candida parapsilosis, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Candida stellatoidea, and Candida tropicalis), Coccidioides immitis, Conidiobolus species, Cryptococcus neoforms, Cunninghamella species, Dermatophytes, Histoplasma capsulatum, Microsporum gypseum, Mucor pusillus, Paracoceidioides brasiliensis, Pseudallescheria boydii, Rhinosporidium seeberi, Pneumocystis carinii, Rhizopus species (e.g., Rhizopus arrhizus, Rhizopus oryzae, and Rhizopus microsporus), Saccharomyces species, Sporothrix schenckii, and classes such as Zygomycetes, Ascomycetes, the Basidiomycetes, Deuteromycetes, and Oomycetes.

In another embodiment, the invention provides methods for preventing, managing, treating or ameliorating cancer, autoimmune, inflammatory or infectious diseases or one or more symptoms thereof, said methods comprising administering to a subject in need thereof a dose of a prophylactically or therapeutically effective amount of one or more scaffolds of the invention in combination with surgery, alone or in further combination with the administration of a standard or experimental chemotherapy, a hormonal therapy, a biological therapy/immunotherapy and/or a radiation therapy. In accordance with these embodiments, the scaffolds of the invention utilized to prevent, manage, treat or ameliorate cancer, autoimmune, inflammatory or infectious diseases or one or more symptoms or one or more symptoms thereof may or may not be conjugated or fused to a moiety (e.g., therapeutic agent or drug).

The invention provides methods for preventing, managing, treating or ameliorating cancer, autoimmune, inflammatory or infectious diseases or one or more symptoms or one or more symptoms thereof, said methods comprising administering to a subject in need thereof one or more scaffolds of the invention in combination with one or more of therapeutic agents that are not cancer therapeutics (a.k.a., non-cancer therapies). Examples of such agents include, but are not limited to, anti-emetic agents, anti-fungal agents, anti-bacterial agents, such as antibiotics, anti-inflammatory agents, and anti-viral agents. Non-limiting examples of anti-emetic agents include metopimazin and metoclopramide. Non-limiting examples of antifungal agents include azole drugs, imidazole, triazoles, polyene, amphotericin and yrimidine. Non-limiting examples of anti-bacterial agents include dactinomycin, bleomycin, erythromycin, penicillin, mithramycin, cephalosporin, imipenem, axtreonam, vancomycin, cycloserine, bacitracin, chloramphenicol, clindamycin, tetracycline, streptomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin, spectinomycin, trimethoprim, norfloxacin, refampin, polymyxin, amphotericin B, nystatin, ketocanazole, isoniazid, metronidazole and pentamidine. Non-limiting examples of antiviral agents include nucleoside analogs (e.g., zidovudine, acyclivir, gangcyclivir, vidarbine, idoxuridine, trifluridine and ribavirin), foscaret, amantadine, rimantadine, saquinavir, indinavir, ritonavir, interferon (“IFN”)-α,β or γ and AZT. Non-limiting examples of anti-inflammatory agents include non-steroidal anti-inflammatory drugs (“NSAIDs”), steroidal anti-inflammatory drugs, beta-agonists, anti-cholingenic agents and methylxanthines.

In another embodiment, the invention comprises compositions capable of inhibiting a cancer cell phenotype. In one embodiment, the cancer cell phenotype is cell growth, cell attachment, loss of cell attachment, decreased receptor expression (such as, for example, but not limited to Eph receptors), increased receptor expression (such as, for example, but not limited to Eph receptors), metastatic potential, cell cycle inhibition, receptor tyrosine kinase activation/inhibition or others.

In one embodiment, the invention comprises compositions capable of treating chronic inflammation. The compositions can be used in the targeting of immune cells for destruction or deactivation. The compositions are useful in targeting activated T cells, dormant T cells, B cells, neutrophils, eosiniphils, basophils, mast cells, or dendritic cells. The compositions may be capable of decreasing or ablating immune cell function.

In another embodiment, the invention comprises compositions capable of inhibiting or reducing angiogenesis. In another embodiment, the angiogenesis is related to tumor growth, rheumatoid arthritis, SLE, Sjogren's syndrome or others.

In another embodiment, the invention comprises compositions useful for treatment of diseases of the gastrointestinal tract. The scaffolds of the invention exhibit a high level of stability under low pH conditions. The stability at low pH suggests that the composition will be suitable for oral administration for a variety of gastrointestinal disorders, such as irritable bowel syndrome, gastroesophageal reflux, intestinal pseudo-obstructions, dumping syndrome, intractable nausea, peptic ulcer, appendicitis, ischemic colitis, ulcerative colitis, gastritis, Helicobacter pylori disease, Crohn's disease, Whipple's disease, celiac sprue, diverticulitis, diverticulosis, dysphagia, hiatus hernia, infections esophageal disorders, hiccups, rumination and others.

The invention further provides combinatorial compositions and methods of using such compositions in the prevention, treatment, reduction, or amelioration of disease or symptoms thereof. The scaffolds of the invention may be combined with conventional therapies suitable for the prevention, treatment, reduction or amelioration of disease or symptoms thereof. Exemplary conventional therapies can be found in the Physician's Desk Reference (56th ed., 2002 and 57th ed., 2003). In some embodiments, scaffolds of the invention may be combined with chemotherapy, radiation therapy, surgery, immunotherapy with a biologic (antibody or peptide), small molecules, or another therapy known in the art. In some embodiments, the combinatorial therapy is administered together. In other embodiments, the combinatorial therapy is administered separately.

The invention also provides methods of diagnosing diseases. The scaffolds of the invention which bind a specific target associated with a disease may be implemented in a method used to diagnose said disease. In one embodiment, the scaffolds of the invention are used in a method to diagnose a disease in a subject, said method comprising obtaining a sample from the subject, contacting the target with the scaffold in said sample under conditions that allow the target:scaffold interaction to form, identifying the target: scaffold complex and thereby detecting the target in the sample.

In some embodiments, the target is an antigen associated with disease. In another embodiment, the target is a cytokine, inflammatory mediator, and intracellular antigen, a self-antigen, a non-self antigen, an intranuclear antigen, a cell-surface antigen, a bacterial antigen, a viral antigen or a fungal antigen. In other embodiments, the disease to be diagnosed is described herein.

The invention also provides methods of imaging specific targets. In one embodiment, scaffolds of the invention conjugated to imaging agents such as green-fluorescent proteins, other fluorescent tags (Cy3, Cy5, Rhodamine and others), biotin, or radionuclides may be used in methods to image the presence, location, or progression of a specific target. In some embodiments, the method of imaging a target comprising a scaffold of the invention is performed in vitro. In other embodiments, the method of imaging a target comprising a scaffold of the invention is performed in vivo. In other embodiments, the method of imaging a target comprising a scaffold of the invention is performed by MRI, PET scanning, X-ray, fluorescence detection or by other detection methods known in the art.

The invention also provides methods of monitoring disease progression, relapse, treatment, or amelioration using the scaffolds of the invention. In one embodiment, methods of monitoring disease progression, relapse, treatment, or amelioration is accomplished by the methods of imaging, diagnosing, or contacting a compound/target with a scaffold of the invention as presented herein.

Kits

Also within the scope of the invention are kits comprising the compositions of the invention (e.g. scaffolds,) and instructions for use. The kit can further contain at least one additional reagent, or one or more additional scaffolds of the invention. Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. This application claims the benefit of priority to U.S. Provisional Application No. 61/323,708 filed Apr. 13, 2010, the entire contents of which are incorporated herein by reference. Additionally, PCT Application No. PCT/US2008/012398, filed on Oct. 10, 2008 and published as International Publication No. WO 2009/058379 is hereby incorporated by reference herein in its entirety for all purposes.

Exemplary Embodiments

-   1. A multimeric scaffold comprising at least two fibronectin type     III (FnIII) scaffolds connected in tandem, wherein each FnIII     scaffold binds a target, and wherein each FnIII scaffold comprises:     -   I. seven beta strand domains designated A, B, C, D, E, F, and G;     -   II. linked to six loop regions, wherein a loop region connects         each beta strand and is designated AB, BC, CD, DE, EF, and FG;     -   wherein each beta strand has at least 50% homology to the         cognate beta strand of a FnIII domain of interest (FOI) and at         least one loop is a non-naturally occurring variant of the         cognate loop in the FOI, and     -   wherein the binding affinity and/or avidity for said target,         and/or a biological activity of the multimeric scaffold is         improved over that of the corresponding monomeric FnIII         scaffolds. -   2. The multimeric scaffold of embodiment 1, wherein the binding     affinity and/or avidity for said target is improved. -   3. The multimeric scaffold of embodiment 1, wherein the binding     affinity and/or avidity for said target, and a biological activity     of the multimeric scaffold are improved. -   4. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 50%     homology to the cognate beta strand domain in any of SEQ ID     NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16). -   5. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 60%     homology to the cognate beta strand domain in any of SEQ ID     NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16). -   6. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 70%     homology to the cognate beta strand domain in any of SEQ ID     NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16). -   7. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 80%     homology to the cognate beta strand domain in any of SEQ ID     NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16). -   8. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 90%     homology to the cognate beta strand domain in any of SEQ ID     NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16). -   9. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 95%     homology to the cognate beta strand domain in any of SEQ ID     NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16)     1-15, 30-48, or 63. -   10. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 98%     homology to the cognate beta strand domain in any of SEQ ID NOs:     1-15, 30-48, or 63. -   11. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 60%     identity to the cognate beta strand domain in any of SEQ ID     NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16). -   12. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 70%     identity to the cognate beta strand domain in any of SEQ ID     NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16). -   13. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 80%     identity to the cognate beta strand domain in any of SEQ ID     NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16). -   14. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 90%     identity to the cognate beta strand domain in any of SEQ ID     NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16). -   15. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 95%     identity to the cognate beta strand domain in any of SEQ ID     NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16). -   16. The multimeric scaffold of embodiment 1, 2 or 3, wherein each     beta strand of at least one of the FnIII scaffolds has at least 98%     identity to the cognate beta strand domain in any of SEQ ID NOs:     1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16). -   17. The multimeric scaffold of any one of the preceeding     embodiments, wherein for at least one FnIII scaffold the A beta     strand domain comprises SEQ ID NO:41, 42, 61, 62, 76, or 77, the B     beta strand comprises SEQ ID NO:43, 63, or 78, the C beta strand     comprises SEQ ID NO:44, 64, or 79, the D beta strand comprises SEQ     ID NO:46, 65, or 80, the E beta strand comprises SEQ ID NO:47, 66,     or 81, the F beta strand comprises SEQ ID NO:48, 67, or 82, and the     G beta strand comprises SEQ ID NO:52, 68, or 83. -   18. The multimeric scaffold of any one of the preceding embodiments,     wherein for at least two of the FnIII scaffolds the A beta strand     comprises SEQ ID NO: 41, 42, 61, 62, 76, or 77, the B beta strand     comprises SEQ ID NO:43, 63, or 78, the C beta strand comprises SEQ     ID NO:44, 64, or 79, the D beta strand comprises SEQ ID NO:46, 65,     or 80, the E beta strand comprises SEQ ID NO:47, 66, or 81, the F     beta strand comprises SEQ ID NO:48, 67, or 82, and the G beta strand     comprises SEQ ID NO:52, 68, or 83. -   19. The multimeric scaffold of embodiment 17 or 18, wherein the AB     loop comprises SEQ ID NO:35, 55, or 70, the CD loop comprises SEQ ID     NO:37, 57, or 72, and the EF loop comprises SEQ ID NO:39, 59, or 74. -   20. The multimeric scaffold of embodiment 17 or 18, wherein the BC     loop comprises SEQ ID NO:36, 56, or 71, the DE loop comprises SEQ ID     NO:38, 58, or 73 and the FG loop comprises SEQ ID NO:39, 59, or 73. -   21. The multimeric scaffold of any one of the preceing embodiments,     wherein for at least one FnIII scaffold the A beta strand domain     comprises SEQ ID NO:41 or 42, the B beta strand comprises SEQ ID     NO:43, the C beta strand comprises SEQ ID NO:45, or 131, the D beta     strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID     NO:47, the F beta strand comprises SEQ ID NO:49 or 51, and the G     beta strand comprises SEQ ID NO:52 or 53. -   22. The multimeric scaffold of any one of the preceding embodiments,     wherein for at least two of the FnIII scaffolds the A beta strand     comprises SEQ ID NO: 41 or 42, the B beta strand comprises SEQ ID     NO:43, the C beta strand comprises SEQ ID NO:45, or 131, the D beta     strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID     NO:47, the F beta strand comprises SEQ ID NO:49 or 51, and the G     beta strand comprises SEQ ID NO:52 or 53. -   23. The multimeric scaffold of any one of embodiments 1-16, 21, or     22, wherein at least one of the FnIII scaffolds comprise the amino     acid sequence:     IEV(X_(AB))_(n)ALITW(X_(BC))_(n)CELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n)YEVSLIC(X_(FG))_(n)KET     FTT, wherein X_(AB), X_(BC) X_(CD), X_(DE), X_(EF), and X_(FG)     represent the amino acid residues present in the AB, BC, CD, DE, EF,     and FG loops, respectively, wherein X₁ represents amino acid residue     A or T, and wherein n=2-26. -   24. The multimeric scaffold of any one of embodiments 1-16, 21, 22     or 23, wherein at least two of the FnIII scaffolds comprise the     amino acid sequence:     IEV(X_(AB))_(n)ALITW(X_(BC))_(n)CELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n)YEVSLIC(X_(FG))_(n)KET     FTT, wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG)     represent the amino acid residues present in the AB, BC, CD, DE, EF,     and FG loops, respectively, wherein X₁ represents amino acid residue     A or T, and wherein n=2-26. -   25. The multimeric scaffold of any one of embodiments 21-24, wherein     the AB loop comprises SEQ ID NO:35, the CD loop comprises SEQ ID     NO:37, and the EF loop comprises SEQ ID NO:39. -   26. The multimeric scaffold of any one of embodiments 21-24, wherein     the BC loop comprises SEQ ID NO: 36, the DE loop comprises SEQ ID     NO:38, and the FG loop comprises SEQ ID NO: 40. -   27. The multimeric scaffold of any one of embodiments 21-25, wherein     for (X_(FG))_(n)n=1, 2, 3, 4, 5, 6, 7, 8, or 9. -   28. The multimeric scaffold of any one of embodiments 1-19, 21, 22,     23, 24, 25, or 27, wherein the BC loop of at least one of the FnIII     scaffolds comprises the sequence: S-X-a-X-b-X-X-X-G, wherein X     represents any amino acid, wherein (a) represents proline or alanine     and wherein (b) represents alanine or glycine. -   29. The multimeric scaffold of any one of embodiments 1-19, 21, 22,     23, 24, 25, or 27, wherein the BC loop of at least one of the FnIII     scaffolds comprises the sequence: S-P-c-X-X-X-X-X-X-T-G, wherein X     represents any amino acid and wherein (c) represents proline, serine     or glycine. -   30. The multimeric scaffold of any one of embodiments 1-19, 21, 22,     23, 24, 25, or 27, wherein the BC loop of at least one of the FnIII     scaffolds comprises the sequence: A-d-P-X-X-X-e-f-X-I-X-G, wherein X     represents any amino acid, wherein (d) represents proline, glutamate     or lysine, wherein (e) represents asparagine or glycine, and     wherein (f) represents serine or glycine. -   31. The multimeric scaffold of any one of embodiments 1-19, 21, 22,     23, 24, 25, or 28-30, wherein the FG loop of at least one of the     FnIII scaffolds comprises the sequence: X-a-X-X-G-X-X-X-b, wherein X     represents any amino acid, wherein (a) represents asparagine,     threonine, or lysine, and wherein (b) represents serine or alanine. -   32. The multimeric scaffold of any one of embodiments 1-19, 21, 22,     23, 24, 25, or 28-30, wherein the FG loop of at least one of the     FnIII scaffolds comprises the sequence: X-a-X-X-X-X-b-N-P-A, wherein     X represents any amino acid, wherein (a) represents asparagine,     threonine or lysine and wherein (b) represents serine or glycine. -   33. The multimeric scaffold of any one of embodiments 1-19, 21, 22,     23, 24, 25, or 28-30, wherein the FG loop of at least one of the     FnIII scaffolds comprises 11 amino acids having a sequence of     X-a-X-X-G-X-X-S-N-P-A, wherein X represents any amino acid, and     wherein (a) represents asparagine, threonine or lysine. -   34. The multimeric scaffold of any one of embodiments 1-19, 21, 22,     23, 24, 25, or 27-33, wherein the DE loop of at least one of the     FnIII scaffolds comprises the sequence: X-X-X-X-X-X, wherein X     represents any amino acid. -   35. The multimeric scaffold of any one of embodiments 1-18, 20, 21,     22, 23, 24, or 26, wherein the AB loop of at least one of the FnIII     scaffolds comprises the sequence: K-X-X-X-X-X-a, wherein X     represents asparagine, aspartic acid, histidine, tyrosine,     isoleucine, valine, leucine, phenylalanine, threonine, alanine,     proline, or serine, and wherein (a) represents serine, threonine,     alanine, or glycine. -   36. The multimeric scaffold of any one of embodiments 1-18, 20, 21,     22, 23, 24, or 26, wherein the AB loop of at least one of the FnIII     scaffolds comprises the sequence: K-X-X-X-X-X-X-X-a, wherein X     represents asparagine, aspartic acid, histidine, tyrosine,     isoleucine, valine, leucine, phenylalanine, threonine, alanine,     proline, or serine, and wherein (a) represents serine, threonine,     alanine, or glycine. -   37. The multimeric scaffold of any one of embodiments 1-18, 20, 21,     22, 23, 24, 26, or 35-36, wherein the CD loop of at least one of the     FnIII scaffolds comprises 7, 8, or 9 residues wherein each residue     in the CD loop is randomized and wherein each residue may be     asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine,     leucine, phenylalanine, threonine, alanine, proline, or serine. -   38. The multimeric scaffold of any one of embodiments 1-18, 20, 21,     22, 23, 24, 26, or 35-37, wherein the EF loop of at least one of the     FnIII scaffolds comprises 8 residues having the sequence     X-b-L-X-P-X-c-X, wherein X represents asparagine, aspartic acid,     histidine, tyrosine, isoleucine, valine, leucine, phenylalanine,     threonine, alanine, proline, or serine, wherein (b) represents     asparagine, lysine, arginine, aspartic acid, glutamic acid, or     glycine, and wherein (c) represents isoleucine, threonine, serine,     valine, alanine, or glycine. -   39. The multimeric scaffold of any one of the preceding embodiments,     wherein the multimeric scaffold comprises at least three FnIII     scaffolds. -   40. The multimeric scaffold of any one of the preceding embodiments,     wherein the multimeric scaffold comprises at least four FnIII     scaffolds. -   41. The multimeric scaffold of any one of the preceding embodiments,     wherein the multimeric scaffold comprises at least five FnIII     scaffolds. -   42. The multimeric scaffold of any one of the preceding embodiments,     wherein the multimeric scaffold comprises at least six FnIII     scaffolds. -   43. The multimeric scaffold of any one of the preceding embodiments,     wherein the multimeric scaffold comprises at least seven FnIII     scaffolds. -   44. The multimeric scaffold of any one of the preceding embodiments,     wherein the multimeric scaffold comprises at least eight FnIII     scaffolds. -   45. The multimeric scaffold of any one of the preceding embodiments,     wherein the multimeric scaffold comprises more than eight FnIII     scaffolds. -   46. The multimeric scaffold of any one of the preceding embodiments,     wherein at least one of the FnIII scaffolds is fused to a     heterologous moiety. -   47. The multimeric scaffold of embodiment 46, wherein the     heterologous moiety is selected from the group consisting of:     polyethylene glycol (PEG), a cytotoxic agent, a radionuclide,     imaging agent, biotin, human serum albumin (HSA) or an FcRn binding     portion thereof, an Fc region of an antibody, a light chain constant     region of an antibody, an albumin binding domain, an IgG molecule,     transferrin, a binding peptide, a non-FnIII scaffold, an epitope     tag, a nucleic acid, a recombinant polypeptide polymer, or a     cytokine. -   48. The multimeric scaffold of any one of the preceding embodiments,     wherein the target is a cell-surface antigen, a soluble antigen, an     immobilized antigen, an immunosilent antigen, an intracellular     antigen, an intranuclear antigen, a self antigen, a non-self     antigen, a cancer antigen, a bacterial antigen, or a viral antigen. -   49. The multimeric scaffold of any one of the preceding embodiments,     wherein the multimeric scaffold is a receptor agonist. -   50. The multimeric scaffold of any one of the preceding embodiments,     wherein the multimeric scaffold binds the target with a K_(D) of     less than 500 μM. -   51. The multimeric scaffold of any one of the preceding embodiments,     wherein the multimeric scaffold binds the target with a K_(D) of     less than 100 μM. -   52. The multimeric scaffold of any one of the preceding embodiments,     wherein two or more FnIII scaffolds bind the same target at the same     epitope. -   53. The multimeric scaffold of any one of the preceding embodiments,     wherein two or more FnIII scaffolds are identical. -   54. The multimeric scaffold of any one of the preceding embodiments,     wherein two or more FnIII scaffolds are not identical. -   55. The multimeric scaffold of any one of the preceding embodiments,     wherein the multimeric scaffold binds at least two different     non-overlapping epitopes on the same target. -   56. The multimeric scaffold of any one of the preceding embodiments,     wherein two or more FnIII scaffolds bind the same target at     non-overlapping epitopes. -   57. The multimeric scaffold of any one of embodiments 1-52, wherein     the FnIII scaffolds bind to the same epitope on two or more copies     of a target molecule on a cell surface. -   58. The multimeric scaffold of any one of embodiments 1-51, or 54,     wherein two or more FnIII scaffolds bind different targets. -   59. The multimeric scaffold of any one of embodiments 1-51, or 54,     wherein the multimeric scaffold binds at least two different     targets. -   60. The multimeric scaffold of any one of the preceding embodiments,     wherein at least two of the FnIII scaffolds are connected in tandem     by a peptide linker. -   61. The multimeric scaffold of any one of the preceding embodiments,     wherein the linker comprises 1 to about 1000 amino acids. -   62. The multimeric scaffold of any one of the preceding embodiments,     wherein the linker comprises 1 to about 50 amino acids. -   63. The multimeric scaffold of any one of the preceding embodiments,     wherein the linker comprises 1 to 25 amino acids. -   64. The multimeric scaffold of any one of the preceding embodiments,     wherein the linker comprises 1 to 15 amino acids. -   65. The multimeric scaffold of any one of the preceding embodiments,     wherein the linker comprises 1 to 5 amino acids -   66. The multimeric scaffold of any one of the preceding embodiments,     wherein the linker is a flexible peptide linker comprising at least     50% glycine residues. -   67. The multimeric scaffold of any one of the preceding embodiments,     wherein the linker sequence comprises one more sequence of the group     consisting of: (G-G-G-S)_(x), (G-G-G-G-S)_(x), (G-G-G-G-S-A)_(x),     (G-A)_(x), (G-G-G-T-P-T)_(x), and (G-G-G-G-S-G-T-G-S-A-M-A-S)_(x)     where x is a positive integer. -   68. The multimeric scaffold of any one of the preceding embodiments,     wherein the linker is a functional moiety. -   69. The multimeric scaffold of any one of the preceding embodiments,     wherein at least one FnIII scaffold is operably linked to an IgG     domain or a full length IgG light or heavy chain. -   70. The multimeric scaffold of embodiment 69, wherein the IgG domain     is selected from the group consisting of:     -   I. an Fc region;     -   II. a CH1 region;     -   III. a CH2 region;     -   IV. a CH3 region;     -   V. a hinge region;     -   VI. a Ckappa region;     -   VII. a Clambda region;     -   VIII. a CH1-hinge-CH2-CH3 region; and     -   IX. a variable region. -   71. An isolated nucleic acid molecule encoding the multimeric     scaffold of any one of the preceeding embodiments. -   72. An expression vector operably linked to the nucleic acid of     embodiment 71. -   73. A host cell comprising the vector of embodiment 72. -   74. A method of producing a multimeric scaffold comprising culturing     the host cell of embodiment 73 under conditions in which the     multimeric scaffold encoded by the nucleic acid molecule is     expressed. -   75. The method of embodiment 74, wherein the expressed multimeric     scaffold is secreted into the culture media. -   76. The method of embodiment 75, further comprising obtaining the     protein from the culture media. -   77. A composition comprising the multimeric scaffold of any one of     embodiments 1-70 in a pharmaceutically acceptable excipient. -   78. A method for treating or inhibiting growth of cancer in a     patient comprising administering an effective amount of the     composition of embodiment 77. -   79. The method of embodiment 78, wherein the cancer is selected from     the group consisting of: squamous cell cancer, small-cell lung     cancer, non-small cell lung cancer, non-Hodgkin's lymphoma,     blastoma, gastrointestinal cancer, renal cancer, ovarian cancer,     liver cancer, stomach cancer, bladder cancer, hepatoma, breast     cancer, colon cancer, colorectal cancer, pancreatic cancer,     endometrial carcinoma, salivary gland carcinoma, kidney cancer,     liver cancer, prostate cancer, vulval cancer, thyroid cancer,     hepatic carcinoma, head and neck cancer, lung cancer,     adenocarcinoma, renal cell carcinoma, or hepatocellular carcinoma. -   80. A method for treating an autoimmune disorder, an inflammatory     disorder, or a respiratory infection in a patient comprising     administering an effective amount of the composition of embodiment     77. -   81. The method of embodiment 80, wherein the respiratory infection     is caused by a virus or bacteria. -   82. The method of embodiment 81, wherein the virus is respiratory     syncytial virus, parainfluenza virus or human metapneumovirus. -   83. The method of embodiment 80, wherein the inflammatory disorder     is asthma, chronic inflammation resulting from chronic viral or     bacterial infections, chronic obstructive pulmonary disease;     encephalitis, inflammatory bowel disease, inflammatory osteolysis,     pulmonary fibrosis, septic shock, undifferentiated arthropathy, or     undifferentiated spondyloarthropathy. -   84. The method of embodiment 80, wherein the autoimmune disorder is     age-related macular degeneration, allograft rejection, ankylosing     spondylitis, antiphospholipid syndrome, arthritis, atherosclerosis,     anaphylactic shock, autoimmune Addison's disease, alopecia greata,     autoimmune diseases of the adrenal gland, autoimmune hemolytic     anemia, autoimmune hepatitis, autoimmune oophoritis, autoimmune     orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous     pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue     immune dysfunction syndrome, chronic inflammatory demyelinating     polyneuropathy, chronic inflammation, Churg-Strauss syndrome,     cicatrical pemphigoid, cold agglutinin disease, corneal and other     tissue transplantation, CREST syndrome, Crohn's disease, cystic     fibrosis, diabetic retinopathies, discoid lupus, endocarditis,     endotoxic shock, essential mixed cryoglobulinemia,     fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,     Guillain-Barre, Hashimoto's thyroiditis, hemangiomas, idiopathic     pulmonary fibrosis, idiopathic thrombocytopenia purpura, IgA     neuropathy, juvenile arthritis, lichen planus, lupus erythematosus,     Meniere's disease, mixed connective tissue disease, multiple     sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia     gravis, neovascular glaucoma, organ ischemia, pemphigus vulgaris,     peritonitis, pernicious anemia, polyarteritis nodosa,     polychrondritis, polyglandular syndromes, polymyalgia rheumatica,     polymyositis and dermatomyositis, primary agammaglobulinemia,     primary biliary cirrhosis, psoriasis, psoriatic arthritis,     Raynauld's phenomenon, Reiter's syndrome, reperfusion injury,     retrolental fibroplasia, rheumatoid arthritis, sarcoidosis,     scleroderma, sepsis, septicemia, Sjogren's syndrome, spinal cord     injury, stiff-man syndrome, systemic lupus erythematosus, takayasu     arteritis, temporal arteritis/giant cell arteritis, thyroid     hyperplasias, ulcerative colitis, uveitis, vasculitides such as     dermatitis herpetiformis vasculitis, vitiligo, and Wegener's     granulomatosis. -   85. A library of diverse fibronectin type III (FnIII) scaffolds     comprising:     -   I. seven beta strand domains designated A, B, C, D, E, F, and G;     -   II. linked to six loop regions, wherein a loop region connects         each beta strand and is designated AB, BC, CD, DE, EF, and FG;     -   wherein each beta strand has at least 50% homology to the         cognate beta strand of a FnIII domain of interest (FOI) and at         least one loop is a non-naturally occurring variant of the         cognate loop in the FOI, and wherein the FG loop is at least one         amino acid shorter than the cognate FG loop in the FOI. -   86. A library of diverse fibronectin type III (FnIII) scaffolds     comprising:     -   I. seven beta strand domains designated A, B, C, D, E, F, and G;     -   II. linked to six loop regions, wherein a loop region connects         each beta strand and is designated AB, BC, CD, DE, EF, and FG;     -   wherein each beta strand domain has at least 50% homology to the         cognate beta strand of a FnIII domain of interest (FOI) and at         least one loop is a non-naturally occurring variant of the         cognate loop in the FOI, and wherein the FG loop consists of no         more than 9 amino acids. -   87. The library of embodiment 85 or 86, wherein each beta strand has     at least 50% homology to SEQ ID NOs:1-34, 54, 69, or the sequences     presented in Table 16 (FIG. 16). -   88. The library of embodiment 85 or 86, wherein each beta strand has     at least 60% homology to any of SEQ ID NOs:1-34, 54, 69, or the     sequences presented in Table 16 (FIG. 16). -   89. The library of embodiment 85 or 86, wherein each beta strand has     at least 70% homology to any of SEQ ID NOs:1-34, 54, 69, or the     sequences presented in Table 16 (FIG. 16). -   90. The library of embodiment 85 or 86, wherein each beta strand has     at least 80% homology to any of SEQ ID NOs:1-34, 54, 69, or the     sequences presented in Table 16 (FIG. 16). -   91. The library of embodiment 85 or 86, wherein each beta strand has     at least 90% homology to any of SEQ ID NOs:1-34, 54, 69, or the     sequences presented in Table 16 (FIG. 16). -   92. The library of embodiment 85 or 86, wherein each beta strand of     the native FnIII domain has at least 95% homology to any of SEQ ID     NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16). -   93. The library of embodiment 85 or 86, wherein each beta strand has     at least 98% homology to any of SEQ ID NOs:1-34, 54, 69, or the     sequences presented in Table 16 (FIG. 16). -   94. The library of embodiment 85 or 86, wherein each beta strand has     at least 60% identity to any of SEQ ID NOs:1-34, 54, 69, or the     sequences presented in Table 16 (FIG. 16). -   95. The library of embodiment 85 or 86, wherein each beta strand has     at least 70% identity to any of SEQ ID NOs:1-34, 54, 69, or the     sequences presented in Table 16 (FIG. 16). -   96. The library of embodiment 85 or 86, wherein each beta strand has     at least 80% identity to any of SEQ ID NOs:1-34, 54, 69, or the     sequences presented in Table 16 (FIG. 16). -   97. The library of embodiment 85 or 86, wherein each beta strand has     at least 90% identity to any of SEQ ID NOs:1-34, 54, 69, or the     sequences presented in Table 16 (FIG. 16). -   98. The library of embodiment 85 or 86, wherein each beta strand has     at least 95% identity to any of SEQ ID NOs:1-34, 54, 69, or the     sequences presented in Table 16 (FIG. 16). -   99. The library of embodiment 85 or 86, wherein each beta strand has     at least 98% identity to any of SEQ ID NOs:1-34, 54, 69, or the     sequences presented in Table 16 (FIG. 16). -   100. The library of embodiment 85 or 86, wherein the A beta strand     domain comprises SEQ ID NO: 41, 42, 61, 62, 76, or 77, the B beta     strand comprises SEQ ID NO:43, 63, or 78, the C beta strand     comprises SEQ ID NO:44, 64, or 79, the D beta strand comprises SEQ     ID NO:46, 65, or 80, the E beta strand comprises SEQ ID NO:47, 66,     or 81, the F beta strand comprises SEQ ID NO:48, 67, or 82, and the     G beta strand comprises SEQ ID NO:52, 68, or 83. -   101. The library of embodiment 100, wherein the AB loop comprises     SEQ ID NO:35, 55, or 70, the CD loop comprises SEQ ID NO:37, 57, or     72, and the EF loop comprises SEQ ID NO:39, 59, or 74. -   102. The library of embodiment 100, wherein the BC loop comprises     SEQ ID NO:36, 56, or 71, the DE loop comprises SEQ ID NO:38, 58, or     73 and the FG loop comprises SEQ ID NO: 40, 60 or 75. -   103. The library of embodiment 85 or 86, wherein the A beta strand     domain comprises SEQ ID NO: 41 or 42, the B beta strand comprises     SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45, or 131, the     D beta strand comprises SEQ ID NO:46, the E beta strand comprises     SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49 or 51, and     the G beta strand comprises SEQ ID NO:52 or 53. [this embodiment     will list SEQ ID NOs for Tn3 beta strands] -   104. The library of embodiment 85 or 86, wherein the FnIII scaffolds     comprise the amino acid sequence:     -   IEV(X_(AB))_(n)ALITW(X_(BC))_(n)CELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n)YEVSLIC(X_(FG))_(n)KET         FTT, wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG)         represent the amino acid residues present in the AB, BC, CD, DE,         EF, and FG loops, respectively, wherein X₁ represents amino acid         residue A or T, and wherein n=3-26 and m=1-9. -   105. The library of embodiment 103 or 104, wherein the AB loop     comprises SEQ ID NO:35, the CD loop comprises SEQ ID NO:37, and the     EF loop comprises SEQ ID NO:39. -   106. The library of embodiment 103 or 104, wherein the BC loop     comprises BC loop comprises SEQ ID NO:36, the DE loop comprises SEQ     ID NO:38, and the FG loop comprises SEQ ID NO:40. -   107. The library of any one of embodiments 85, 86, 100, 101, 103, or     105, wherein the amino acid sequence of the BC loop of the FnIII     scaffold comprises the sequence of: S-X-a-X-b-X-X-X-G, wherein X     represents any amino acid, wherein (a) represents proline or alanine     and wherein (b) represents alanine or glycine. -   108. The library of any one of embodiments 85-, 86, 100, 101, 103,     or 105, wherein the amino acid sequence of the BC loop of the FnIII     scaffold comprises the sequence of: S-P-c-X-X-X-X-X-X-T-G, wherein X     represents any amino acid and wherein (c) represents proline, serine     or glycine. -   109. The library of any one of embodiments 85, 86, 100, 101, 103, or     105, wherein the amino acid sequence of the BC loop of the FnIII     scaffold comprises the sequence: A-d-P-X-X-X-e-f-X-I-X-G, wherein X     represents any amino acid, wherein (d) represents proline, glutamate     or lysine, wherein (e) represents asparagine or glycine, and     wherein (f) represents serine or glycine. -   110. The library of any one of embodiments 85, 86, 100, 101, 103, or     105, wherein the amino acid sequence of the FG loop of the FnIII     scaffold comprises the sequence: X-X-X-X-X-X-X-X-X, wherein X     represents any amino acid. -   111. The library of any one of embodiments 85, 86, 100, 101, 103, or     105, wherein the amino acid sequence of the FG loop of the FnIII     scaffold comprises the sequence: X-a-X-X-G-X-X-X-b, wherein X     represents any amino acid, wherein (a) represents asparagine,     threonine, or lysine, and wherein (b) represents serine or alanine. -   112. The library of any one of embodiments 85, 86, 100, 101, 103, or     105, wherein the amino acid sequence of the DE loop of the FnIII     scaffold comprises the sequence: X-X-X-X-X-X, wherein X represents     any amino acid. -   113. The library of any one of embodiments 85, 86, 100, 100, 103, or     106, wherein the amino acid sequence of the AB loop of the FnIII     scaffold comprises the sequence: K-X-X-X-X-X-a, wherein X represents     asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine,     leucine, phenylalanine, threonine, alanine, proline, or serine, and     wherein (a) represents serine, threonine, alanine, or glycine. -   114. The library of any one of embodiments 85-, 86, 100, 100, 103,     or 106, wherein the amino acid sequence of sad AB loop of the FnIII     scaffold comprises the sequence: K-X-X-X-X-X-X-X-a, wherein X     represents asparagine, aspartic acid, histidine, tyrosine,     isoleucine, valine, leucine, phenylalanine, threonine, alanine,     proline, or serine, and wherein (a) represents serine, threonine,     alanine, or glycine. -   115. The library of any one of embodiments 85-, 86, 100, 100, 103,     or 106, wherein the amino acid sequence of the CD loop of the FnIII     scaffold comprises 7, 8, or 9 residues wherein each residue in the     CD loop is randomized and wherein each residue may be asparagine,     aspartic acid, histidine, tyrosine, isoleucine, valine, leucine,     phenylalanine, threonine, alanine, proline, or serine. -   116. The library of any one of embodiments 85-, 86, 100, 100, 103,     or 106, wherein the amino acid sequence of the EF loop of the FnIII     scaffold comprises the sequence: X-b-L-X-P-X-c-X, wherein X     represents asparagine, aspartic acid, histidine, tyrosine,     isoleucine, valine, leucine, phenylalanine, threonine, alanine,     proline, or serine, wherein (b) represents asparagine, lysine,     arginine, aspartic acid, glutamic acid, or glycine, and wherein (c)     represents isoleucine, threonine, serine, valine, alanine, or     glycine. -   117. The library of any one of embodiments 85-116, wherein the     library is displayed on the surface of a ribosome, bacteriophage,     virus, bacteria, yeast, or mammalian cell. -   118. A method for identifying a fibronectin type III (FnIII)     scaffold from the library of any one of embodiments 85-117, wherein     the FnIII scaffold has increased protein stability as compared to an     the FOI, and binds a target, comprising:     -   I. contacting the target ligand with the library of any one of         embodiments 85-117 under conditions suitable for forming a         scaffold:target ligand complex;     -   II. obtaining from the complex, the scaffold that binds the         target ligand;     -   III. determining if the stability of the scaffold obtained in         step (II) is greater than that of the FOI. -   119. The method of embodiment 118, further comprising randomizing at     least one loop of the scaffold obtained in step (II) and repeating     steps (I) and (II) using the further randomized scaffold. -   120. A method for obtaining a fibronectin type III (FnIII) scaffold     variant having increased stability as compared to an FnIII scaffold     of interest (FOI), comprising: engineering a variant of the FOI,     wherein the FG loop of the variant comprises the deletion of at     least 1 amino acid, wherein the variant exhibits an increased     stability as compared to the FOI. -   121. The method of embodiment 120, wherein the length and sequence     of the FG loop is determined prior to engineering by aligning the     amino acid sequence of the FOI with the amino acid sequence of at     least one native FnIII domain. -   122. The method of embodiment 120, wherein the length and sequence     of the FG loop is determined prior to engineering by modeling the     three dimensional structure of at least one native FnIII domain on     the amino acid sequence of the FOI. -   123. The method of any one of embodiments 118-122, wherein the     protein stability is measured by melting temperature, differential     scanning calorimetry (DSC), circular dichroism (CD), polyacrylamide     gel electrophoresis (PAGE), protease resistance, isothermal     calorimetry (ITC), nuclear magnetic resonance (NMR), internal     fluorescence, and/or biological activity. -   124. The method of any one of embodiments 118-123, wherein the     stability is increased by at least 10% in Cm as compared to the FOI. -   125. The method of any one of embodiments 118-124, wherein the     stability is increased by at least 20% in Cm as compared to the FOI. -   126. The method of any one of embodiments 118-125, wherein the     stability is measured by urea denaturation. -   127. The method of any one of embodiments 118-125, wherein the     stability is measured by guanidine denaturation. -   128. The method of any one of embodiments 118-127, wherein the     scaffold exhibits a decrease of at least 10% in protease sensitivity     as compared to the FOI. -   129. The method of any one of embodiments 118-128, wherein the     scaffold exhibits an increased melting temperature as compared to     the FOI. -   130. The recombinant scaffold of embodiment 118-129, wherein the     melting temperature is increased by at least 2° C. as compared to     the FOI. -   131. The method of any one of embodiments 118-130, wherein the FOI     comprises any of SEQ ID NOs:1-34, 54, 69, or the sequences presented     in Table 16 (FIG. 16). -   132. The method of any one of embodiments 118-131, wherein the FOI     comprises SEQ ID NO: 1. -   133. The method of any one of embodiments 118-131, wherein the FOI     SEQ ID NO: 54 or 69. -   134. A fibronectin type III scaffold having increased protein     stability produced by the method of any one of embodiments 118-133,     wherein the scaffold exhibits an increased stability of: (a) of at     least 10% in Cm as measured in a urea denaturation experiment; (b)     of at least 10% in Cm as measured in a guanidine denaturation     experiment; (c) of at least 10% in protease sensitivity; or (d)     increased melting temperature, as compared to that of the FOI.

TABLE 1 Sequences and SEQ ID Nos of molecular components to assemble representative scaffolds on the invention: SEQ Name/Brief Description Sequence ID NO Tn3 IEVKDVTDTTALITWFKPLAEIDGCELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDTEYEVSL 1 ICRRGDMSSNPAKETFTT (cys residues of disulfide bond are underlined) SS3 IEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSAGNLKPDTEYCVSL 2 ISRRGDMSSNPAKECFTT (cys residues of disulfide bond are underlined) Tn3 + SS3 IEVKDVTDTTALITWFKPLAEIDGCELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDTEYCVSL 3 ICRRGDMSSNPAKECFTT (cys residues of disulfide bonds are underlined) 3rd FnIII of tenascin C RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPD 4 (w/N-term aa) TEYEVSLISRRGDMSSNPAKETFTT (underlined A beta strand residues may be removed) 10th FnIII of LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITV 5 fibronectin YAVTGRGDSPASSKPISINYRT 3rd FnIII of PTVDQVDDTSIVVRWSRPQAPITGYRIVYSPSVEGSSTELNLPETANSVTLSDLQPGVQYNITIY 6 fibronectin AVEENQESTPVVIQQET 6th FnIII of PYNTEVTETTIVITWTPAPRIGFKLGVRPSQGGEAPREVTSDSGSIVVSGLTPGVEYVYTIQVLR 7 fibronectin DGQERDAPIVNKVVT FnIII from growth PPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWKMMDPILTTSV 8 hormone R PVYSLKVDKEYEVRVRSKQRNSGNYGEFSEVLYVTLP FnIII from β PPSLNVTKDGDSYSLRWETMKMRYEHIDHTFEIQYRKDTATWKDSKTETLQNAHSMALPALEPST 9 common R RYWARVRVRTSRTGYNGIWSEWSEARSWDTE FnIII from IL-5R PPVNFTIKVTGLAQVLLQWKPNPDQEQRNVNLEYQVKINAPKEDDYETRITESKIVTILHKGFSA 10 SVRTILQNDHSLLASSWASAELHA 29^(th) FnIII from LSVTDVTTSSLRLNWEAPPGAFDSFLLRFGVPSPSTLEPHPRPLLQRELMVPGTRHSAVLRDLRS 11 Tenascin XB GTLYSLTLYGLRGPHKADSIQGTART 31^(st) FnIII from LRALNLTEGFAVLHWKPPQNPVDTYDIQVTAPGAPPLQAETPGSAVDYPLHDLVLHTNYTATVRG 12 Tenascin XB LRGPNLTSPASITFTT 32^(nd) FnIII from LEAKEVTPRTALLTWTEPPVRPAGYLLSFHTPGGQTQEILLPGGITSHQLLGLFPSTSYNARLQA 13 Tenascin XB MWGQSLLPPVSTSFTT Truncated 3rd FnIII IEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDTEYEVSL 14 of tenascin C ISRRGDMSSNPAKETFTT FnIII-growth hormone R PKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQLFYTRRNTQEWTQEWKECPDYVSAGENSCYFN 15 SSFTSIWIPYCIKLTSNGGTVDEKCFSV FnIII from PTPR-F PSGFPQNLHVTGLTTSTTELAWDPPVLAERNGRIISYTVVFRDINSQQELQNITTDTRFTLTGLK 16 PDTTYDIKVRAWTSKGSGPLSPSIQSRTMPVE FnIII from PTPR-F PKPPIDLVVTETTATSVTLTWDSGNSEPVTYYGIQYRAAGTEGPFQEVDGVATTRYSIGGLSPFS 17 EYAFRVLAVNSIGRGPPSEAVRARTGE FnIII from collagen LSPPRNLRISNVGSNSARLTWDPTSRQINGYRIVYNNADGTEINEVEVDPTIIFPLKGLTPLTEY 18 type XIV TIAIFSIYDEGQSEPLTGVFTT 3rd FnIII of tenascin IEKVKDVTDTTALITWFKPLAEIDGIQLTYGIKDVPGDRTTINLTEDEVQYSIGNLKPDTEYEVS 19 C-charge variant SLISRRGDMSSNPAKQTFTT Archaeoglobus fulgidus PAISNVRVSDVTNSSATIRWDVSLAANNRVLFSTNSDLSSPQWSAWDNSTDSPMITLSGLSAGTA 20 DSM 4304 NCBI Acc. #: YYFSVYSFRPDNASLYSNSSIMSFTT NC_000917 Staphylothermus marinus SEPQNLKATAGNNNITLTWDPPIDDGGCRIVEYRIYRGTNNNNLEYYASVNGSTTTFIDKNIVYS 21 F1 NCBI Acc. #: QTYYYKVSAVNNIVEGPKSNTASATPTSS NC_009033 Sulfolobus acidocaldarius PPPKPVIRFAQAGNNSISLSWYDTNTSGYYIQWWSSIDNNKSTINVGNVSSYLFINLTNGVTYYF 22 DSM 639 NCBI Acc. #: RIIPYNQAGNGTSSDIISLTPGAV NC_007181 1st FnIII Sulfolobus acidocaldarius PDSPSVKVIVGDRNATVIWSKPYNGGFPILGYYLTVKTDNSSYTINVGNVSKYTLTNLTPEVLYE 23 DSM 639 NCBI Acc. #: VMVVAYNKLGNSSPGIVNFVALTT NC_007181 2nd FnIII Sulfolobus acidocaldarius LTTASISVSVYKKVNGVLISWNKTENTTYYNLLISDKKGKIIVNITTTNTSYFAYIPYGIYNVTI 24 DSM 639 NCBI Acc. #: RATNQVGTNSTSFPIVFYIPPFI NC_007181 3rd FnIII Sulfolobus acidocaldarius PLVKFSIGNNSILNLKWNNVTGATFYLVYVNTTLIANVTTDSYSLNLTPGFHVIRVVAANPIYNS 25 DSM 639 NCBI Acc. #: SPASLGILIQQHSVTSSIT NC_007181 4th FnIII Sulfolobus solfataricus PLPPKITSYSAGNESVTLGWNPVRLSSGYEIIYWNNMGFNSSINVGNVTSYTVTGLKDGITYYFE 26 P2 NCBI Acc. #: VLAYNSIGYSSPSSIIALTPASV NC_002754 1st FnIII Sulfolobus solfataricus PNPPQLVSVKYGNDNVTLNWLPPTFSGGYLLLGYYVIVKNENSMVSSHFVNSTSLTISNLTPNVT 27 P2 NCBI Acc. #: YNVFIYAVNKLGNSSPLVLTVVPITKA NC_002754 2nd FnIII Sulfolobus solfataricus PITKASVFAFITKLGNGILVNWTTSFPANTLELYNPNGNLISQIAAIKGNSSYLFRVPQGNYTLV 28 P2 NCBI Acc. #: IIASNSAGVSKYVYQVVYYL NC_002754 3rd FnIII Sulfolobus solfataricus PPASPQVLSIGFGNNLYISWNNEANVITYLVYVNNSLVYEGPSNSIVTNISNGTYLVKVIGVNPA 29 P2 NCBI Acc. #: GSSSPGIAVIHYTGDYVT NC_002754 4th FnIII Sulfolobus tokodaii str. PPKPQIASIASGNETITVKWYDTNASGYYITWSNFSQKVTINGNVTSYTIKHLKDGVTYYIQIVP 30 7 NCBI Acc. #: YNSLGNGTPSDIISATPSSV NC_003106 1st FnIII Sulfolobus tokodaii str. PNPPIIKVKIGNLNATLTWYDTFNGGYPIEGYYLYVNGKGINVGNITSYVLTNLTAGELYTIELI 31 7 NCBI Acc. #: AYNKIGNSSISSVSFIAASKA NC_003106 2nd FnIII Sulfolobus tokodaii str. ASKANLTVTVYKKINGFLVSWNSTSKAKYILTVSKENVVLLNVSTTNTSYFVKPFGVYNISLEAV 32 7 NCBI Acc. #: NIVGITKYAFILIYYIQ NC_003106 3rd FnIII Sulfolobus tokodaii str. PASPTVNWSITLNTVSLNWSKVSGAEYYLIYDNGKLITNTTNTAFTFNLTIGQNEIEVYAANAYY 33 7 NCBI Acc. #: KSAPYIINDVRNYIVV NC_003106 4th FnIII 14^(th) FnIII of ARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLY 34 fibronectin TLNDNARSSPVVIDAST 3^(rd) FnIII of tenascin KDVTDTT 35 C, AB loop 3^(rd) FnIII of tenascin FKPLAEIDG 36 C, BC loop 3^(rd) FnIII of tenascin KDVPGDR 37 C, CD loop 3^(rd) FnIII of tenascin TEDENQ 38 C, DE loop 3^(rd) FnIII of tenascin GNLKPDTE 39 C, EF loop 3^(rd) FnIII of tenascin RRGDMSSNPA 40 C, FG loop 3^(rd) FnIII of tenascin RLDAPSQIEV 41 C, beta strand A 3^(rd) FnIII of tenascin IEV 42 C, beta strand A N-terminal truncation 3^(rd) FnIII of tenascin ALITW 43 C, beta strand B 3^(rd) FnIII of tenascin IELTYGI 44 C, beta strand C 3^(rd) FnIII of tenascin CELTYGI 45 C, beta strand C (Tn3) 3^(rd) FnIII of tenascin TTIDL 46 C, beta strand D 3^(rd) FnIII of tenascin YSI 47 C, beta strand E 3^(rd) FnIII of tenascin YEVSLIS 48 C, beta strand F 3^(rd) FnIII of tenascin YEVSLIC 49 C, beta strand F (Tn3) 3^(rd) FnIII of tenascin YCVSLIS 50 C, beta strand F (SS3) 3^(rd) FnIII of tenascin YCVSLIC 51 C, beta strand F (Tn3 + SS3) 3^(rd) FnIII of tenascin KETFTT 52 C, beta strand G 3^(rd) FnIII of tenascin KECFTT 53 C, beta strand G (SS3 & Tn3 + SS3) WT 10FnIII of fibronectin VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPG 54 (w/N-term aa) VDYTITVYAVTGRGDSPASSKPISINYRT (underlined A beta strand residues may be removed) WT 10FnIII of fibronectin, VAATPTS 55 AB loop WT 10FnIII of fibronectin, DAPAVTVRY 56 BC loop WT 10FnIII of fibronectin, TGGNSPV 57 CD loop WT 10FnIII of fibronectin, PGSKST 58 DE loop WT 10FnIII of fibronectin, SGLKPGVD 59 EF loop WT 10FnIII of fibronectin, VTGRGDSPASSKPI 60 FG loop WT 10FnIII of fibronectin, VSDVPRDLEV 61 beta strand A WT 10FnIII of fibronectin, LEV 62 beta strand A N-terminal truncation WT 10FnIII of fibronectin, LLISW 63 beta strand B WT 10FnIII of fibronectin, YRITYGE 64 beta strand C WT 10FnIII of fibronectin, GEFTV 65 beta strand D WT 10FnIII of fibronectin, ATI 66 beta strand E WT 10FnIII of fibronectin, YTITVYA 67 beta strand F WT 10FnIII of fibronectin, SINYRT 68 beta strand G WT 14FnIII of fibronectin, VSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTD 69 (w/N-term aa) YKIYLYTLNDNARSSPVVIDAST (underlined A beta strand residues may be removed) WT 14FnIII of fibronectin, TDATETT 70 AB loop WT 14FnIII of fibronectin, RTKTETITG 71 BC loop WT 14FnIII of fibronectin, ANGQTP 72 CD loop WT 14FnIII of fibronectin, KPDVRS 73 DE loop WT 14FnIII of fibronectin, TGLQPGTD 74 EF loop WT 14FnIII of fibronectin, LNDNARSSPV 75 FG loop WT 14FnIII of fibronectin, SPPRRARV 76 Beta strand A WT 14FnIII of fibronectin, ARV 77 Beta strand A N-terminal truncation WT 14FnIII of fibronectin, ITISW 78 Beta strand B WT 14FnIII of fibronectin, FQVDAVP 79 Beta strand C WT 14FnIII of fibronectin, IQRTI 80 Beta strand D WT 14FnIII of fibronectin, YTI 81 Beta strand E WT 14FnIII of fibronectin, YKIYLYT 82 Beta strand F WT 14FnIII of fibronectin, VIDAST 83 Beta strand G Fc region with hinge EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD 84 GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CH1-hinge-Fc region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL 85 SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK Kappa light chain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST 86 YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Lambda light chain QPKAAPSVLTFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKY 87 AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC Linker region 1 GGGGSGGGGSGGGGSA 88 Linker region 2 GGGGSGGGGSGTGSAMASGGGGSA 89 Linker region from C1

RLDAPGQ 90 (G-G-G-G-S) units are in bold; natural tenascin C sequence underlined Linker region from

RLDAPGQ 91 C2 and C8 (G-G-G-G-S) units are in bold; natural tenascin C sequence underlined Linker region from C3

RLDAPGQ 92 (G-G-G-G-S) units are in bold; natural tenascin C sequence underlined Linker region from C4

RLDAPGQ 93 (G-G-G-G-S) units are in bold; natural tenascin C sequence underlined Linker region from C5

RLDAPGQ 94 natural tenascin C sequence underlined Linker region from C6

RLDAPGQ 95 (G-G-G-G-S) units are in bold; natural tenascin C sequence underlined Linker region from C7

RLDAPGQ 96 (G-G-G-G-S) units are in bold; natural tenascin C sequence underlined Tenascin-consensus FnIII LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGT 256 (w/N-term aa) EYTVSIYGVKGGHRSNPLSAEFTT (underlined A beta strand residues may be removed) Tenascin-consensus FnIII SEVTEDS 257 AB loop Tenascin-consensus FnIII TAPDAAFDS 258 BC loop Tenascin-consensus FnIII SEKVGEA 259 CD loop Tenascin-consensus FnIII PGSERS 260 DE loop Tenascin-consensus FnIII TGLKPGTE 261 EF loop Tenascin-consensus FnIII VKGGHRSNPL 262 FG loop Tenascin-consensus FnIII LPAPKNLVV 263 Beta strand A Tenascin-consensus FnIII LVV 264 Beta strand A N-terminal truncation Tenascin-consensus FnIII LRLSW 265 Beta strand B Tenascin-consensus FnIII FLIQYQE 266 Beta strand C Tenascin-consensus FnIII INLTV 267 Beta strand D Tenascin-consensus FnIII YDL 268 Beta strand E Tenascin-consensus FnIII YTVSIYG 269 Beta strand F Tenascin-consensus FnIII SAEFTT 270 Beta strand G

EXAMPLES

The invention is now described with reference to the following examples. These examples are illustrative only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.

Example 1 Design of Various Multivalent Tn3 Formats

Multivalent formats of the Tn3 scaffold have been designed. The multivalent formats contain one or more Tn3 modules fused to themselves, fused to other protein motifs that can oligomerize, or fused to themselves and to other protein motifs that can oligomerize are shown in FIG. 1. In each case, the resulting molecular entity contains at least 2 Tn3 modules. The polypeptide linkers connecting the Tn3 modules to each other or to other protein motifs can be structured or unstructured and with or without a function. Three exemplary classes of multivalent Tn3 scaffold proteins are specifically provided: (i) linear (L) multivalent proteins containing Tn3 modules fused to each other via a polypeptide linker; (ii) antibody-like (Ig) multivalent proteins containing one or more linearly fused Tn3 modules fused to the light and heavy chains of an antibody or antibody fragment and (iii) Fc-containing multivalent proteins containing one or more linearly fused Tn3 modules fused to an antibody Fc region (FIG. 1).

Example 2 Expression and Purification of Multivalent TRAIL R2-specific Tn3-containing Proteins

A series of eight multivalent Tn3-module containing scaffold proteins (also referred to as “Tn3 proteins” or “Tn3 scaffolds”) with binding specificity for human TRAIL R2 were prepared. Examples were prepared from each of the three multivalent formats described in Example 1, and all of these proteins presented 2 or more of the TRAIL R2-binding Tn3 module A1 (clone 1E11, G6 or 1C12). For several TRAIL R2-specific multivalent Tn3 protein, a corresponding control Tn3 protein (clone DE a Tn3 domain specific for the Synagis® antibody) that did not bind TRAIL R2 was also generated, differing only in the sequence and binding specificity of the component Tn3 modules. Tn3 clone D1 is a Tn3 protein wherein the BC, DE, and FG loops of a 1E11 clone are replaced with alternative loops with sequences corresponding to SEQ ID NO: 99, 38, and 107, respectively (see TABLE 4). Sequence identity numbers of the multivalent Tn3 protein constructs that were expressed are shown in TABLE 2, and all the possible constructs are represented schematically in TABLE 3 and FIG. 2. The loop sequences for the clones are provided in TABLE 4.

TABLE 2 Names, formats, valencies, and specificities of expressed Tn3- containing proteins Name Format Number of (clone) type SEQ ID NO Tn3 modules Specificity A1(1E11) Monomer 134 1 TRAIL R2 A2(1E11) L 139 2 TRAIL R2 A3(1E11) L 140 4 TRAIL R2 A4(1E11) L 141 6 TRAIL R2 A5(1E11) L 142 8 TRAIL R2 A5(G6) L 145 8 TRAIL R2 A6(1E11) Fc 151 2 TRAIL R2 A7(1E11) Fc 164 4 TRAIL R2 A8(1E11) Fc 165 8 TRAIL R2 A9(1C12) Ig 154 (HC), 4 TRAIL R2 154 (LC) A9(1E11) Ig 158 (HC), 4 TRAIL R2 159 (LC) B1(D1) Monomer 180 1 non TRAIL R2-binding control of A1 B2(D1) L not expressed 2 non TRAIL R2-binding control of A2 B3(D1) L 146 4 non TRAIL R2-binding control of A3 B4(D1) L 147 6 non TRAIL R2-binding control of A4 B5(D1) L 148 8 non TRAIL R2-binding control of A5 B6(D1) Fc 181 2 non TRAIL R2-binding control of A6 B7(D1) Fc not expressed 4 non TRAIL R2-binding control of A7 B8(D1) Fc not expressed 8 non TRAIL R2-binding control of A8 B9(D1) Ig 182 (HC), 4 non TRAIL 183 (LC) R2-binding control of A9 L = linear Tn3 fusions, Fc = Fc-Tn3 fusions, Ig = antibody-like Tn3 fusions

TABLE 3 Schematic Representation of Tn3 Scaffold Constructs Construct Components Tn3 Module (Tn3) IEV(X_(AB))_(n)ALITW(X_(BC))_(n)CELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n)YEVSLIC(X_(FG))_(n)KETFTT X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG )represent the amino acid residues present in the AB, BC, CD, DE, EF, and FG loops, respectively where n = 2-26, X₁ represents amino acid residue A or T. Gly-Ser linker GGGGS module, (G₄S)_(n) The (G₄S)n module wherein n = 1 is shown above where n = 1-7 Poly-Histidine Tag HHHHHHHH (

) An optional component of the constructs detailed below-useful for purification Name Construct Overview A1 or B1 A(Tn3)GGGTLG

A2 or B2 S(G₄S)₁A(Tn3)(G₄S)₃A(Tn3)(G₄S)₂GTL

A3 or B3 S(G₄S)₁A(Tn3)(G₄S)₃A(Tn3)(G₄S)₃A(Tn3)(G₄S)₂GTLG

A4 or B4 S(G₄S)₁A(Tn3)(G₄S)₃A(Tn3)(G₄S)₃A(Tn3)(G₄S)₃A(Tn3)(G₄S)₂GTGSAMAS(G₄S)₁A(Tn3) (G₄S)₃A(Tn3)(G₄S)₂GTLG

A5 or B5 S(G₄S)₁A(Tn3)(G₄S)₃A(Tn3)(G₄S)₃A(Tn3)(G₄S)₃A(Tn3)(G₄S)₂GTGSAMAS(G₄S)₁A(Tn3) (G₄S)₃A(Tn3)(G₄S)₃A(Tn3)(G₄S)₃A(Tn3)(G₄S)₂GTLG

A6 or B6 (Tn3)GAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK A7 or B7 AMAS(G₄S)₁A(Tn3)(G₄S)₃A(Tn3)(G₄S)₂GTGAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK A8 or B8 AMAS(G₄S)₁A(Tn3)(G₄S)₃A(Tn3)(G₄S)₃A(Tn3)(G₄S)₃A(Tn3)(G₄S)₂GTGAEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK A9 or B9 heavy chain SQ(Tn3)GGGTPTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ constant region SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK fusion DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK A9 or B9 light chain SQ(Tn3)GGGTPTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD constant region SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVKTSFNRGEC fusion M13 or 79 A(Tn3)GGGTLG

C1 A(Tn3)A(G₄S)₁RLDAPGQ(Tn3)GGGTLG

C2 A(Tn3)(G₄S)₃RLDAPGQ(Tn3)GGGTLG

C3 A(Tn3)(G₄S)₃RLDAPGQ(Tn3)GGGTLG

C4 A(Tn3)(G₄S)₇RLDAPGQ(Tn3)GGGTLG

C5 A(Tn3)TRLDAPGQ(Tn3)GGGTLG

C6 A(Tn3)(G₄S)₁RLDAPGQ(Tn3)GGGTLG

C7 A(Tn3)(G₄S)₂RLDAPGQ(Tn3)GGGTLG

C8 A(Tn3)(G₄S)₃RLDAPGQ(Tn3)GGGTLG

TABLE 4 Loop Sequences of Tn3 Clones Used in These Studies AB Loop BC Loop CD Loop DE Loop EF Loop FG Loop Clone (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) 1E11† KDVTDTT AKPWVDPPPLWG KDVPGDR QQKHTA GNLKPDTE FDPYGAKSNPA (NO: 35) (NO: 97) (NO: 37) (NO: 102) (NO: 39) (NO: 106) D1 KDVTDTT SPGERIWMFTG KDVPGDR TEDENQ GNLKPDTE PNYERISNPA (NO: 35) (NO: 99) (NO: 37) (NO: 38) (NO: 39) (NO: 107) G6† KDVTDTT AKPWVDPPPLWG KDVPGDR QQKHTA GNLKPDTE FDPYGMRSKPA (NO: 35) (NO: 97) (NO: 37) (NO: 102) (NO: 39) (NO: 108) 1C12† KDVTDTT AKPEKWDGSIYG KDVPGDR NSRHTA GNLKPDTE FTPYGAKSNPA (NO: 35) (NO: 98) (NO: 37) (NO: 103) (NO: 39) (NO: 109) M13 KDVTDTT HDAFGYDFG KDVPGDR PDHFHN GNLKPDTE ANDHGFDSNPA (NO: 35) (NO: 100) (NO: 37) (NO: 104) (NO: 39) (NO: 110) 79 KDVTDTT IPPHNADSSIIG KDVPGDR YDVAFD GNLKPDTE DTFYGFDSNPA (NO: 35) (NO: 101) (NO: 37) (NO: 105) (NO: 39) (NO: 111) G3† KDVTDTT AKPEKWDGPPLW KDVPGDR NSRHTA GNLKPDTE FTPYGAKSNPA (NO: 35) (NO: 168) (NO: 37) (NO: 103) (NO: 39) (NO: 109) C4† KDVTDTT AKPWVDPPPLWG KDVPGDR QQKHTA GNLKPDTE FDPYNKRNVPA (NO: 35) (NO: 97) (NO: 37) (NO: 102) (NO: 39) (NO: 169) F4† KDVTDTT AKPWVDPPPLWG KDVPGDR QQKHTA GNLKPDTE FDPYGLKSRPA (NO: 35) (NO: 97) (NO: 37) (NO: 102) (NO: 39) (NO: 170) F4mod1† KDVTDTT AKPWVDPPPLWG KDVPGDR QQKHTA GNLKPDTE FDPYGLKSRPA (NO: 35) (NO: 9897 (NO: 37) (NO: 102) (NO: 39) (NO: 170) F4mod12 KDVTDTT AKPWVDPPPLWG KDVPGDR QQKHNQ GNLKPDTE FDPYGLKSRPA (NO: 35) (NO: 97) (NO: 37) (NO: 179) (NO: 39) (NO: 170) †Clones comprising a C beta strand having the sequence CELAYGI (SEQ ID NO: 131), all other clones comprise a C beta strand having the sequence CELTYGI (SEQ ID NO: 45)

Preparation of Expression Constructs:

Enzymes used were from New England Biolabs (Ipswich, Mass.), DNA purification kits were from Qiagen (Germantown, Md.), and DNA primers were from IDT (Coralville, Iowa). Preparation of expression constructs encoding 2 or more linearly fused Tn3 modules was as follows. The DNA encoding a TRAIL R2-specific Tn3 module (e.g., 1E11, SEQ ID NO: 134; G6, SEQ ID NO: 138; etc.) was amplified by PCR with the primers “Tn3 gly4serl module forward” (SEQ ID NO: 112) and “Tn3 gly4ser2 module reverse” (SEQ ID NO: 113) (TABLE 5).

After cleanup of the PCR product, the amplified DNA was divided in two, with one half digested with BpmI, and the other half digested with AcuI. The digested samples were purified using a PCR cleanup kit and ligated with T4 DNA ligase to make a DNA product encoding two Tn3 modules (A2). This material was purified by agarose gel electrophoresis and again split into two. Digestion with NcoI and KpnI followed by ligation into NcoI/KpnI digested pSec-oppA(L25M) (described in WO 2009/058379 A2, Example 18) yielded the bacterial expression construct for protein A2. Ligation of undigested product into pCR 2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) provided genetic material for generation of higher order fusions. To make a DNA fragment encoding four Tn3 modules (A3), the TOPO cloned A2 DNA was PCR amplified with primers “module amp forward” (SEQ ID NO: 114) and “module amp reverse” (SEQ ID NO: 115) (TABLE 5), purified, and split in two for digestion with AcuI or BpmI. The rest of the process for making the A3 expression construct was the same as that used for making the A2 construct, wherein the DNA encoding A3 was assembled from A2 building blocks. Again, concurrent cloning of assembled A3 DNA into pCR 2.1-TOPO provided genetic material for generation of higher order fusions.

For preparation of A4 and A5 bacterial expression constructs, an adapter module was introduced at the 3′ end of the multi-Tn3 coding sequence within the A3 expression construct. To do this, the A3 expression vector was first digested with KpnI and EcoRI, and the excised fragment was replaced with a duplex cassette containing the oligonucleotides “insert BamHI in pSec forward” (SEQ ID NO: 116) and “insert BamHI in pSec reverse” (SEQ ID NO: 117) (TABLE 5). PCR amplification of A2 and A3 sequences from the corresponding pCR 2.1 TOPO constructs was performed with the primers “module insert BamHI forward” (SEQ ID NO: 118) and “module amp reverse” (SEQ ID NO: 115) (TABLE 5). Amplified products were double digested with BamHI/KpnI, and cloned into similarly digested A3 expression construct.

Proteins A6-A9 were expressed by transient transfection of 293F cells, as described in Example 16 of WO 2009/058379 A2. Briefly, expression vectors were generated by PCR amplifying the Tn3 module (or modules) from the bacterial expression constructs, and cloning these into in house vectors encoding the Fc region, the kappa light chain constant region and/or the CHI-hinge-CH2-CH3 heavy chain constant regions for expression of Fc fusion or antibody proteins. For protein A9, a Tn3 module replaces the antibody variable regions in the human IgG1 heavy chain and kappa light chain. The primers that add compatible NheI and KasI sites for making Fc fusions of the tandem constructs are shown in TABLE 5.

TABLE 5 Primer Sequences Used in the Construction of Multivalent Tn3 Proteins Sequence Name Sequence SEQ ID NO Tn3 gly4ser1 module GGCGCTAGGCTGAGTAGGTCCTGGAGTGCGGCCATGGCCAGCGG 112 forward GGGCGGAGGGAGTGCCATTGAAGTGAAAGATGTGACCGATACC Tn3 gly4ser2 module CCTCAGCCGATCACCACCTGAAGGCTACGCAGGTACCGCTACCG 113 reverse CCACCTCCGCTCCCACCGCCACCGGTGGTAAAGGTTTC Module amp forward GGCGCTAGGCTGAGTAGGTCCTGGAGTGCGG 114 Module amp reverse CCTCAGCCGATCACCACCTGAAGGCTACGCAGG 115 Module insert GGGATCCGCTACGGGCCACTCGATCGAGGTCCGTGCTGATCGAG 116 BamHI in pSec CGATCGGTACCCTGGGCCATCATCATCATCATCACCACCACTGA forward G Module insert AATTCTCAGTGGTGGTGATGATGATGATGATGGCCCAGGGTACC 117 BamHI in pSec GATCGCTCGATCAGCACGGACCTCGATCGAGTGGCCCGTAGCGG reverse ATCCCGTAC Module insert GGCGCTAGGCTGAGTAGGTCCTGGGGATCCGCCATGGCCAGC 118 BamHI forward Module insert NheI GGCGCTAGGCTGAGTAGGTCCTGGCTAGCTGCCATGGCCAGC 119 forward Module insert KasI CCTCAGCCGATCACCACCTGAAGGCGGCGCCGGTACC 120 reverse

Expression and Purification of Proteins:

Monovalent or linear Tn3 proteins were expressed in BL21(DE3) E. coli (EMD/Novagen, Gibbstown, N.J.) and the His-tagged proteins were purified from the culture media using Ni NTA Superflow resin (Qiagen). Surprisingly, despite large differences in the molecular weights, all of these constructs expressed at medium to high levels in E. coli and were efficiently secreted into the media (TABLE 6 and FIG. 3).

To express Fc fusion and antibody-like proteins (A6-A9), 293F cells were transiently transfected with the appropriate expression constructs. Harvests of supernatant were performed on days 6 and 10 and the protein was purified by protein A affinity chromatography.

All purified proteins were analysed by SDS-PAGE on NuPage Novex 4-12% bis tris gels in MES buffer without reducing agent, and were visualized using SimplyBlue SafeStain (Invitrogen, Carlsbad, Calif.). Size exclusion chromatography was also used to analyze purified proteins, and where necessary, aggregated material was removed on either a Superdex 75 10/300GL or Superdex 200 10/300GL column (GE Healthcare, Piscataway, N.J.), to a final level below 10% of total protein. An Acrodisc unit with a Mustang E membrane (Pall Corporation, Port Washington, N.Y.) was used as indicated by the manufacturer to remove endotoxin from bacterially expressed protein preparations.

TABLE 6 Yield After Purification of Representative Multivalent Tn3 Protein Formats Protein (Clone) Yield (mg/L) A1 (1E11) 400 A2 (1E11) 300 A3 (1E11) 135 A4 (1E11) 90 A5 (1E11) 40

Example 3 TRAIL R2Binding Affinity for Mono- and Polyvalent Tn3 Proteins

To measure the effect of Tn3 valency on binding affinity for a series of TRAIL R2-specific Tn3 proteins, a competition ELISA experiment was performed. A 96-well NUNC MaxiSorp plate (Thermo Fisher, Rochester, N.Y.), was coated with A9(1C12) (SEQ ID NO: 154+SEQ ID NO: 145) a TRAIL R2 specific scaffold in an antibody-like format, in PBS at 2 μg/ml overnight at 4° C. Plates were blocked with PBS 0.1% Tween 20+10 mg/ml BSA. Dilutions of A1 (1E11 monomer), and linear format A2 (1E11 bivalent) or A3 (1E11 tetravalent) multimeric scaffolds were incubated on the coated plate with 0.75 nM of biotinylated TRAIL R2-Fc for two hours at room temperature in PBS 0.1% Tween 20+1 mg/ml BSA, washed. Bound biotinylated TRAIL R2Fc was detected with streptavidin HRP, TMB, and neutralized with acid. Absorbance was read at 450 nm. Data is shown in FIG. 4. Binding affinities (IC₅₀) are shown in TABLE 7 and were calculated as the concentration of competing protein required to reduce maximal binding of biotinylated TRAIL R2-Fc by 50%.

The IC₅₀ values for A2 and A3 were at least 30-fold lower than those of the monomer A1 and are at the limit of this assay (i.e., approx. equal to the concentration of biotinylated TRAIL R2-Fc) Binding of biotinylated TRAIL R2-Fc to immobilized TRAIL R2-specific Tn3 was displaced by the TRAIL R2 binding constructs.

Relative to the monomeric A1 protein, the bi- and tetravalent A2 and A3 proteins bound TRAIL R2-Fc with 30-40-fold higher affinity, which is an indication that the multiple Tn3 modules retain their binding activity and contribute to higher affinity through an avidity effect. The true difference in affinity between mono- and bi- or tetravalent Tn3 proteins may be greater than 30-40-fold given the IC₅₀ values for A2 and A3 were approximately equal to the concentration of biotinylated TRAIL R2-Fc used in the assay (0.75 nM).

TABLE 7 IC₅₀ Values for the Inhibition of Binding of TRAIL R2-Fc to immobilized TRAIL R2 Binding A9(1C12) Tn3 Protein Clone Valency IC₅₀ (nM) A1 (1E11) 1 16 A2 (1E11) 2 0.5 A3 (1E11) 4 0.4

Example 4 Flow Cytometry for Confirmation of Cell Binding

Flow cytometry was used to confirm the specificity of binding of a multivalent TRAIL R2-specific Tn3 protein to endogenous TRAIL R2 expressed on the cell surface of H2122 cells. Adherent H2122 cells (a non-small cell lung cancer adenocarcinoma cell line), were detached from tissue culture flasks using Accutase (Innovative Cell Technologies, San Diego, Calif.). Cells were rinsed with complete medium (RPMI 1640 medium supplemented with 10% FBS) and resuspended in PBS/2% FBS at approximately 2×10⁶ cells/mL. Tn3 protein A9(1E11) (SEQ ID NO: 158+SEQ ID NO: 159), a tetravalent antibody-like format multimeric scaffold, or the format-matched control Tn3 protein B9 (clone D1), were prepared at 40 nM concentrations in PBS/2% FBS.

Cells were plated on 96 well U-bottom plates at 75 μl per well, and protein samples were added at 25 μl per well (to a final concentration of 10 nM). The plate was incubated at 4° C. for approximately 1 hour, then washed 3 times with PBS/2% FBS. Anti-human IgG Alexa Fluor 488 conjugated secondary antibody added was added (100 μl/well), and the plate was incubated at 4° C. for approximately 30 minutes and washed as described above. Cells were resuspended in 100 μl of PBS/2% FBS, and flow cytometry analysis was performed using a BD LSR II cytometer (BD Biosciences, San Jose, Calif.). A shift (increase) in fluorescently labeled H2122 cells when incubated with the TRAIL R2 specific Tn3 protein relative to control confirmed that the TRAIL R2 specific Tn3 protein could bind to cellular TRAIL R2 (FIG. 5).

Example 5 Effect of Valency and Format on Apoptosis of H2122 Cells by TRAIL R2-specific Tn3 Proteins

Apoptotic cell death can be induced in cancer cells lines by crosslinking of cell surface TRAIL R2. This effect can be determined in cell assays that measure the number of viable cells. To this end, lung carcinoma cell lines H2122 cells were plated in 96 well plates at a density of 10,000 cells/well in 75 μl of complete medium (RPMI 1640 medium supplemented with 10% FBS). Following overnight incubation at 37° C., media was supplemented with 25 μl of additional media containing a serial dilution of TRAIL R2-specific (clone 1E11) or negative control (clone D1) Tn3 proteins. All treatments were performed in duplicate wells. Commercially available TRAIL ligand (Chemicon/Millipore, Billerica, Mass.) was used as a positive control for TRAIL receptor-induced cell death. After 72 hrs, the CellTiter-Glo kit from Promega (Madison, Wis.) was used according to the manufacturer's instructions to assay ATP levels, which is a measure of the number of viable cells in the culture. Assay luminescence was measured on an Envision Plate reader (PerkinElmer, Waltham, Mass.). Inhibition of cell viability was determined by dividing the luminescence values for treated cells by the average luminescence for untreated viable cells. Dose response plots of inhibition vs compound concentration were generated, and cell killing potency (EC₅₀) was determined as the concentration of protein required to inhibit 50% of the cell viability.

To test the effect of valency on the proapoptotic activity of multivalent TRAIL R2-specific Tn3 proteins, H2122 cells were treated with the monovalent Tn3 protein A1 (clone 1E11), and the series of linearly fused Tn3 proteins A2-A5 (each clone 1E11) which contain 2, 4, 6 or 8 Tn3 modules. While the mono- and bivalent Tn3 proteins showed no or negligible killing activity, proteins containing 4, 6 and 8 Tn3 modules potently inhibited H2122 cell viability, with potency increasing as a function of valency (FIG. 6A; TABLE 8). Protein A3 (tetravalent) had a similar potency to TRAIL, the natural TRAIL R2 ligand, while proteins A4 (hexavalent) and A5 (octavalent) were 1-2 logs more potent. It is clear from this assay that for a given molecular format, cell killing improves with higher valency, up to a point where the assay can no longer discriminate.

TABLE 8 EC₅₀ Values for Killing of H2122 by Multivalent Constructs Clone EC₅₀ (nM) Maximum Inhibition % A3 (1E11) 0.013 91 A4 (1E11) 0.0009 97 A5 (1E11) 0.0006 97 human TRAIL 0.027 98

To demonstrate that inhibition of cell viability is dependent on TRAIL R2 binding, 100 pM of protein A5 (clone G6) (i.e., 167× the EC₅₀) was incubated with H2122 cells in the presence of soluble TRAIL R2-Fc protein. Dose dependent repression of cell killing by soluble TRAIL R2-Fc is an indication that cell killing is dependent on protein A5 binding to cell surface TRAIL R2 (FIG. 6B). Similar results were seen with protein A5 comprising clone 1E11 loops (data not shown).

In addition to the number of binding modules, the activity of multivalent Tn3 proteins may also be affected by the molecular format used to present the individual binding units. To test the effect of molecular format on activity, H2122 cells were treated with different TRAIL R2-specific Tn3 proteins presenting the same number of Tn3 binding modules. The ability of the tetravalent proteins A3, A7 and A9 (each clone 1E11) to induce killing of H2122 cells was tested in the cell viability assay, as was the pair of octavalent Tn3 proteins A5 and A8 (each clone 1E11). Inactive mono- and bivalent proteins were included as negative controls, and TRAIL as a positive control (FIG. 7; TABLE 9 and TABLE 10). In FIG. 7A, for the three constructs tested with a valency of four, it is apparent that A3 (linear format) and A7 (Fc-fusion format) are similar in their cell killing activity and are more potent in killing H2122 cells than A9 (antibody-like fusion format). This clearly shows that the spatial orientation of Tn3 modules can have a considerable effect on bioactivity, wherein A3 is approximately 150-fold more potent than A9 protein in inhibiting 112122 cell viability (TABLE 9). FIG. 7B shows that both formats of octavalent TRAIL R2-binding Tn3 proteins, A5 (linear) and A8 (Fc-fusion), have similar efficacy in inhibiting the viability of H2122 cells. The EC₅₀ data for these constructs is shown in TABLE 9. The ability to fine tune affinity, valency, and spatial orientation affords great flexibility in terms of the ability to precisely engineer a desired therapeutic outcome.

TABLE 9 EC₅₀ Values for Killing of H2122 by Multivalent Constructs with a Valency of Four Clone EC₅₀ (nM) Maximum Inhibition % A9 (1E11) 1.98 80 A7 (1E11) 0.02 88 A3 (1E11) 0.013 91 human TRAIL 0.027 98

TABLE 10 EC₅₀ Values for Killing of H2122 by Multivalent Constructs with a Valency of Eight Maximum Clone EC₅₀ (nM) Inhibition % A5 (1E11) 0.0006 97 A8 (1E11) 0.0002 98 human TRAIL 0.027 98

Example 6 Dose Dependent Cell Killing in the Cell Lines Colo205 and Jurkat

To demonstrate that multivalent TRAIL R2-specific Tn3 proteins could kill cancer cell lines other than H2122, other TRAIL R2 expressing cell lines were also tested. The colorectal adenocarcinoma cell line Colo205 (FIG. 8A) and Jurkat T cell leukemia line (FIG. 8B) were tested for their ability to be killed by proteins A3 (tetravalent, linear format) (SEQ ID NO: 143) and A5 (octavalent, linear format) (SEQ ID NO: 145) (each clone G6). Each cell line was incubated with A3, A5, the positive control TRAIL, or a negative control protein B5 (SEQ ID NO: 148) which does not bind TRAIL R2, and the cell viability assay was performed as described for 142122. In each of these cell lines, A5 shows extremely potent inhibition of cell viability. The lower valency A3 protein also induces cell killing, albeit with lower potency than A5. Thus, the higher valency construct shows greater activity. As expected, TRAIL could also inhibit cell viability, but not octavalent negative control protein B5, which does not bind TRAIL R2.

TABLE 11 EC₅₀ Values for Killing of Colo205 by Linear Tandem Constructs Clone EC₅₀ (nM) Maximum Inhibition % A3 (G6) 0.04 97 A5 (G6) 0.0005 100 human TRAIL 0.08 100

TABLE 12 EC₅₀ Values for Killing of Jurkat cells by Linear Tandem Constructs Maximum Clone EC₅₀ (nM) Inhibition % A3 (G6) 0.05 83 A5 (G6) 0.0001 100 human TRAIL 0.009 99 Cells were analyzed by the CellTiter-Glo assay as in Example 5.

Example 7 Design, Expression, and Activity of Mouse CD40L-Specific Bivalent Tandem Scaffolds

Bivalent murine CD40L-specific Tn3 proteins (TABLE 13) were prepared by fusing a pair of identical Tn3 modules. M13 is a Tn3 protein that specifically binds Murine CD40L. The M13 sequence corresponds to the sequence of Tn3 wherein the sequences of the BC, DE, and FG loops are replaced with alternative loops with sequences corresponding to SEQ ID NOs: 100, 104, and 110, respectively (see TABLE 4). Linkers containing 1 (Construct Cl(M13)), 3 (Construct C2(M13)), 5 (Construct C3(M13)), or 7 (Construct C4(M13)) copies of the Gly₄Ser (GS) unit were used resulting in total linker lengths between 13 and 43 amino acids (see FIG. 9A and TABLE 3).

TABLE 13 Names, valencies, and specificities of expressed Tn3-containing proteins Number of Linker Name (clone) Tn3 modules length Specificity M13 (M13) 1 N/A Murine CD40L C1 (M13) 2 13 Murine CD40L C2 (M13) 2 23 Murine CD40L C3 (M13) 2 33 Murine CD40L C4 (M13) 2 43 Murine CD40L

Briefly, the expression constructs were generated as follows: Fragment A was generated by PCR amplification of Murine CD40L binder pSec-M13 cloned in the pSec-oppA(L25M) vector described in Example 1 with a primer specific for the pSec vector upstream of the Tn3 gene and primer “1-3 GS linker reverse” (SEQ ID NO: 123) (see TABLE 14 for sequences of Tn3 specific primers used). Fragments B1 GS and B3GS were generated by PCR amplification of the same template with primers “1 GS linker” (SEQ ID NO: 121) or “3 Glinker” (SEQ ID NO: 122), respectively, and a primer specific for the pSec vector downstream of the Tn3 gene. Upon gel-purification of the fragments, Fragment A and B1GS or Fragment A and B3GS were mixed, and the tandem constructs were generated by overlap PCR in a PCR reaction with the two pSec vector specific primers. The products were digested with NcoI and KpnI and cloned back into the pSec-oppA(L25M) vector as described in Example 1, yielding the two constructs: C1(M13) and C2(M13). In order to generate the 5 and 7 GS linker constructs, linker inserts generated by PCR amplification of the oligonucleotides “5 GSLinker” (SEQ ID NO: 124) and “7 GSLinker” (SEQ ID NO: 125), respectively, with primers “GS L Amp forward” (SEQ ID NO: 126) and “GS L Amp reverse” (SEQ ID NO: 127) were digested with PstI and XmaI and cloned into a vector fragment generated by cutting pSecM13-1GS-M13 with PstI and XmaI yielding the constructs C3(M13) and C4(M13).

TABLE 14 Primer sequences used in the construction of Tandem bivalent MuCD40L specific constructs Sequence Name Sequence SEQ ID NO 1 GSLinker AAAGAAACCTTTACCACTGCAGGTGGCGGAGGTTCACGCTTG 121 GATGCCCCCGGGCAGATTGAAGTGAAAGATGTGACCGAT 3 GSLinker AAAGAAACCTTTACCACTGCAGGTGGCGGAGGTTCAGGTGGC 122 GGAGGTTCAGGTGGCGGAGGTTCACGCTTGGATGCCCCCGGG CAGATTGAAGTGAAAGATGTGACCGAT 1-3 GSlinker reverse CTGCAGTGGTAAAGGTTTCTTTCG 123 5 GSLinker AAAGAAACCTTTACCACTGCAGGTGGCGGGGGTAGCGGTGGC 124 GGAGGTTCTGGTGGCGGGGGTAGCGGTGGCGGAGGTTCTGGT GGCGGGGGTAGCCGCTTGGATGCCCCCGGGCA 7 GSLinker AAAGAAACCTTTACCACTGCAGGTGGCGGGGGTAGCGGTGGC 125 GGAGGTTCTGGTGGCGGGGGTAGCGGTGGCGGAGGTTCTGGT GGCGGGGGTAGCGGTGGCGGAGGTTCTGGTGGCGGGGGTAGC CGCTTGGATGCCCCCGGGCA GS L Amp forward AAAGAAACCTTTACCCACTGCAGGT 126 GS L Amp reverse TTCAATCTGCCCGGGGGCATCCAA 127

Monovalent and bivalent tandem constructs comprising identical Tn3 scaffolds were recombinantly expressed and purified from E. coli as described in Example 2. FIG. 9B depicts an SDS-PAGE analysis of the purified protein preps under reducing and non-reducing conditions.

In order to test the binding efficiencies of the bivalent tandem M13-M13 constructs and compare them to the monovalent M13 scaffold, their competitive inhibition of Murine CD40L binding to Murine CD40 receptor immobilized on a biosensor chip was tested.

Briefly, a fragment of the Murine CD40 receptor in the form of a chimeric fusion with the Fc region of IgG1 was immobilized onto a GLC chip (Bio-Rad) at a density of about 3000 response units. For competition binding assays, 3-fold serial dilutions of monovalent M13 or the M13 tandem bivalent constructs with different linker length were incubated for 20 min with a fixed concentration of E. coli produced recombinant Murine CD40L (0.5 μg/ml) in PBS containing 0.1% (v/v) Tween-20 and 0.5 mg/mL BSA. These samples were then injected over the GLC chip at a flow rate of 30 μL/min for 300 seconds and the level of bound CD40L was recorded at a fixed time point within the sensorgram and compared to the corresponding level of bound protein in the absence of any competitor. After each binding measurement, residual CD40L was desorbed from the chip surface by injecting 10 mM glycine-HCl (pH 2.0). Non-specific binding effects were corrected by subtracting sensorgrams from interspots of the chip. IC₅₀ values corresponding to the concentrations of Tn3 constructs required to displace 50% of murine CD40L were calculated using GraphPad Prism.

As shown in FIG. 9C, the half maximal inhibitory concentration (IC₅₀) for the M13 monomer was 71 nM while the IC₅₀ for the bivalent tandem construct C1 (M13) was 29 nM. Similar IC₅₀ values of 5 or 6 nM were obtained for the bivalent constructs containing longer linkers (constructs C2(M13), C3(M13) and C4(M13), respectively). Due to the concentration of CD40L used in the assay, this is at the lower limit of IC₅₀s that can be observed in this assay. The bivalent constructs all had a lower IC₅₀ value compared to the monovalent construct, indicating enhanced binding activity of the bivalent tandem constructs compared to a single M13 Tn3 module. The linker length in these bivalent constructs exhibits some effect on assay potency, with the shortest linker length construct having intermediate potency, while those constructs with linkers of 23 or more amino acids are equivalent in this assay.

To test the activity of the bivalent tandem Tn3 constructs in a cell based activity and compare them to the monovalent M13 scaffold, inhibition of Murine CD40L-induced CD86 expression on B-cells was tested. As a control, the commercially available anti-murine CD40L specific antibody (MR1) was tested in parallel.

The assay utilizes PBMC prepared from blood from healthy volunteers. Briefly, freshly drawn blood was collected in BD Vacutainer® CPT™ Cell Preparation Tube with heparin. After centrifugation, the cell layer containing PBMCs was collected and washed twice with PBS and once with RPMI 1640 medium. The cells were resuspended in complete RPMI 1640 medium (supplemented with 10% heat-inactivated fetal bovine serum, 1% P/S) at a concentration of 5×10⁶ cells/ml.

The murine CD40L-expressing Th2 cell line D10.G4.1 was washed and resuspended in complete RPMI 160 medium at a concentration of 1×10⁶ cells/ml.

M13, M13-M13 tandem bivalent constructs C₁-C₄, or MR1 antibody (BioLegend Cat. No: 106508) were serially diluted (1:3) in complete RPMI 1640 medium. A 50 μl sample of each dilution was added to wells in a 96 well U bottom tissue culture plate. Each well then received 50 μl of D10.G4.1 cells (5×10⁴), and after mixing, plates were incubated at 37° C. for 1 hr. 100 μl of resuspended PBMC (5×10⁵ cells) were then added to each well and incubated at 37° C. for 20-24 hrs.

PBMC were collected and stained with APC-anti-human CD86 (BD bioscience, Cat #555660) and FITC-anti-human CD19 (BD bioscience, Cat #555412) in FACS buffer (PBS pH 7.4, 1% BSA, 0.1% sodium azide) at 4° C. for 30 min in the dark. After two washes in FACS buffer, samples were then analyzed by FACS LSRII (Becton Dickinson). CD86 expression on CD19 gated B cells was evaluated. The analysis of CD86 expression as a function of test protein was performed using GraphPad Prism software.

As shown in FIG. 9D, the bivalent M13-M13 tandem constructs all inhibited CD86 expression with an IC₅₀ of 100 to 200 pM, comparable to the IC₅₀ of the MR1 antibody (100 pM) and about 3 logs more potent than the M13 monovalent scaffold itself. In contrast to the biochemical assay, no effect of linker length was observed in this cell based assay, and bivalent constructs with linkers ranging from 13 to 43 amino acids in length all show equivalent enhanced potency relative to the monovalent protein.

Example 8 Expression of Bi-Specific Tandem Scaffolds

To generate bispecific Tn3 constructs with specificity for TRAIL R2 and Human CD40L (HuCD40L), two Tn3 modules, one with specificity for TRAIL R2 (clone 1E11) and one with specificity for human CD40L (clone 79), were fused together with variable length linkers separating the two modules (TABLE3 and TABLE 15). The sequence of the clone 79 protein (SEQ ID NO: 184) corresponds to the sequence of a Tn3 module wherein the BC, DE, and EF loops have been replaced with alternative loops corresponding to SEQ ID NOs: 101, 105, and 111, respectively. Expression constructs for the tandem bispecific scaffolds containing linkers with 1 and 3 Gly₄Ser (GS) repeats (constructs C6 and C8, respectively) were generated as described in Example 7 except that plasmids carrying the Tn3 variants A1 and 79 were used initially as PCR templates. Construct C5 (containing a short linker derived from the natural sequence linking the second and third FnIII domains in human tenascin C, which may be considered part of the A beta strand of the third FnIII domain although it is not required for scaffold binding) and construct C7 were generated in a similar way to C6 and C8, using the additional primers listed in TABLE 17, except that “0 GSlinker reverse” was used in place of “1-3 GSLinker reverse” for C5.

TABLE 15 Names, valencies, and specificities of expressed Tn3-containing proteins Number of Tn3 Linker Name modules length Specificity A1 (1E11) 1 N/A TRAIL R2 79 (79) 1 N/A HuCD40L C5 (1E11 & 79) 2  8 TRAIL R2 + HuCD40L C6 (1E11 & 79) 2 13 TRAIL R2 + HuCD40L C7 (1E11 & 79) 2 18 TRAIL R2 + HuCD40L C8 (1E11 & 79) 2 23 TRAIL R2 + HuCD40L

TABLE 17 Additional Primer sequences used in the construction of bispecific tandem constructs Sequence Name Sequence SEQ ID NO 0 GSLinker AAAGAAACCTTTACCACCACGCGTTTGGATGCCCCCGGGCAGATTG 128 AAGTGAAAGATGTGACCGAT 0 GSlinker reverse CGTGGTGGTAAAGGTTTCTTTCG 129 2 GSLinker AAAGAAACCTTTACCACTGCAGGTGGCGGAGGTTCAGGTGGCGGAG 130 GTTCACGCTTGGATGCCCCCGGGCAGATTGAAGTGAAAGATGTGAC CGAT

Monovalent as well as tandem bispecific Tn3 scaffolds were recombinantly expressed in E. coli media as described in Example 2. Expression levels of the soluble constructs were analyzed using SDS-PAGE. FIG. 10 demonstrates acceptable expression levels for the constructs tested.

Example 9 Specific Binding of BiSpecific Tandem Scaffolds

To measure the binding of bispecific Tn3 constructs to CD40L and TRAIL R2, a capture ELISA assay was employed. Briefly, 8×His-tagged protein constructs: A1, 79, C5, C6, C7 or C8 (see TABLE 15 for details) were captured from E. coli media onto anti-His antibody coated wells as follows. A 96-well MaxiSorb plate was coated with Qiagen anti-His antibody at 2 μg/ml overnight. The coated plate was blocked with PBS containing 0.1% v/v Tween-20 and 4% w/v skim milk powder (PBST 4% milk) for 1.5 hours. The coated plate was washed with PBST and diluted bacterial media (diluted 30-fold) containing soluble expressed proteins was added and plates were incubated at room temperature for 2 hours. After washing with PBST, wells containing the captured constructs were incubated for 1.5 hours with varying concentrations of either biotinylated TRAIL R2 (FIG. 11A) or a complex generated by preincubation of E. coli produced His-tagged HuCD40L with biotinylated anti-His antibody (FIG. 11B). After washing with PBST, bound TRAIL R2 or HuCD40L/anti-H is antibody complex was detected with streptavidin-horseradish peroxidase (RPN1231V; GE Healthcare; 1000×working dilution) for 20 min., washing with PBST, and detecting colorimetrically by addition of TMB substrate (Pierce). The absorbance was read at 450 nm.

Binding of the bispecific tandem TRAIL R2-HuCD40L-specific scaffolds to TRAIL R2, and binding of the bispecific tandem TRAIL R2-HuCD40L-specific scaffolds to HuCD40L are depicted in FIG. 11A and FIG. 11B, respectively. Bispecific tandem scaffolds, designated C5 to C8, comprising a TRAIL R2 specific Tn3 domain fused to a HuCD40L specific Tn3 domain bound TRAIL R2 and HuCD40L; however, the monomeric/monospecific Tn3 constructs A1 and 79 bound either TRAIL R2 or HuCD40L according to their known specificities but not both targets.

Simultaneous binding of tandem TRAIL R2-HuCD40L-specific constructs to TRAIL R2 and HuCD40L was determined using an AlphaScreen™ assay. Dilutions of E. coli media containing proteins A1, 79, C5, C6, C7 and C8 were incubated with 10 nM TRAIL R2-Fc fusion protein, 50 nM biotinylated HuCD40L (produced in E. coli), streptavidin AlphaScreen donor beads (0.02 mg/ml) and Protein A AlphaScreen acceptor beads (0.02 mg/ml) in PBS+0.01% Tween+0.1% BSA. Samples were incubated 1 h in the dark prior to reading in a PerkinElmer Envision reader. The donor bead population was excited with a laser at 680 nm causing the release of singlet oxygen. Singlet oxygen has a limited lifetime allowing it to travel up to 200 nm by diffusion before falling back to ground state. Singlet oxygen excites the acceptor beads causing light emission between 520-620 nm which is measured by the Envision reader. Only when donor and acceptor beads are in proximity is a signal generated. Thus, an increase in signal is observed when the two bead types are brought together by molecules interacting with the two targets simultaneously. In the absence of binding to either target no signal should be detected.

As shown in FIG. 12, the tandem bispecific constructs simultaneously bound TRAIL R2 and HuCD40L generating a strong AlphaScreen signal; however, the monovalent Tn3 scaffolds, A1 and 79, did not generate a signal indicating they could not bring donor and acceptor beads in proximity by simultaneously binding both targets.

Example 10 Increased Stability of Tn3 Scaffolds Having 9 Amino Acid Length FG Loop

To measure the effect of FG loop length on Tn3 stability, unfolding of six

HuCD40L-specific Tn3 scaffolds by guanidine hydrochloride (GuHCl) at pH 7.0 was assessed by intrinsic tryptophan fluorescence. These Tn3 monomeric scaffolds contained FG loop lengths of 9, 10 or 11 amino acids. Samples of 0.05 mg/mL Tn3 scaffold containing different concentrations of guanidine hydrochloride were prepared in 50 mM sodium phosphate pH 7.0. Fluorescence emission spectra were acquired on a Horiba Fluoromax-4 spectrofluorometer at an excitation wavelength of 280 nm. Relative fluorescence emission intensity at 360 nm was plotted as a function of GuHCl concentration for each protein. Each scaffold contained unique BC, DE, and FG loop sequences. Clones A3 (SEQ ID NO:185; note that the A3 monomeric scaffold in this example is distinct from the construct designated A3 as provided in Table 3), 71 (SEQ ID NO: 186), 79 (SEQ ID NO: 184), 127 (SEQ ID NO: 187), 252 (SEQ ID NO: 188), and 230 (SEQ ID NO: 189) were more than 50% unfolded in 3.0M GuHCl at pH 7.0, which is the GuHCl concentration required to effect 50% unfolding (C_(m)) of parental Tn3. C_(m) values for clones A3, 79, 127, 252, and 230 were 2.2M, 2.7M, 2.4M, 2.7M, 2.4M, respectively. The FG loop lengths for these clones is 11, 11, 11, 10 and 11 amino acids respectively, while the FG loop length for parental Tn3 is 10 amino acids. Surprisingly, clone 71, the only variant having an FG loop length of 9 amino acids, exhibited a Cm of 4.2M, a significantly higher stability than parental Tn3 scaffold or the other five variants tested. Results are shown in FIG. 13.

To determine whether the enhanced stability of Tn3 clone 71 was intrinsic to its sequence, or a consequence of the shortened FG loops length, this clone and two additional monomeric Tn3 scaffold proteins, (A6 (SEQ ID NO: 190; note that the A6 monomeric scaffold in this example is distinct from the construct designated A6 as provided in Table 3) and P1C01 (SEQ ID NO: 191)), with an FG loop length of 9 amino acids (but different BC, DE and FG loop sequences) were analyzed by differential scanning calorimetry (DSC) and compared to the parental Tn3 scaffold which contains an FG loop that is 10 amino acids long. Tn3 protein samples at 1 mg/mL in PBS pH 7.2 were analyzed. In all cases, the midpoint of thermal unfolding was higher for clones with the 9 residue FG loops as compared to parental (WT) Tn3, which has a 10 residue FG loop. Thermal unfolding was reversible, or partially reversible (clone A6) as evidenced by superimposable thermograms when the same sample was cooled and reheated. As shown in FIG. 14, the melting temperature (T_(m)) for parental Tn3 was 72.1° C., for P1C01 the Tm was 75.2° C., for A6 the T. was 77.5° C., and for 71 the T. was 74.4° C.

These findings were corroborated by testing the same Tn3 protein variants in a guanidine hydrochloride stability experiment. Unfolding of parental (WT) Tn3, P1C01, A6, and 71 by guanidine hydrochloride (GuHCl) at pH 7.0 was assessed by intrinsic tryptophan fluorescence as described above. As shown in FIG. 15, in agreement with the DSC data in FIG. 14, Tn3 clones A6, 71, and P1C01 all have midpoints of unfolding at significantly higher GuHCl concentrations than parental (WT) Tn3 scaffold, indicating the stability of Tn3 proteins having FG loops that are 9 amino acids in length, i.e. shorter than that in the parental Tn3 scaffold, is enhanced.

Example 11 Stability Analysis of FG Loop Length

As described above, preliminary analysis indicated that Tn3 molecules having an FG loop length of 9 residues are significantly more stable than those having longer FG loops. In these studies, we conducted stability analysis on a set of random Tn3s to assess the effect of FG loop length on thermal stability.

A Tn3 library was subcloned into the pSEC expression vector. This library codes for Tn3s with BC, DE, and FG loops of varying sequence as well as varying but defined length. The FG loop, which is the focus of these studies, can be 9, 10, or 11 residues long. The BC loop may be 9, 11, or 12 residues long. The DE loop in this library has a fixed length of 6 residues. The subcloned library was used to transform DH5α competent cells, from which a plasmid pool was purified and used to transform BL21(DE3) cells. BL21 colonies were sequenced to identify 96 clones which coded for full-length Tn3s. The final 96 clones were grown in a 96 deep-well plate at a 500 μl scale using standard Magic Media expression (37° C. shaking for 24 hours post-innoculation) and analyzed on SDS-PAGE. 29 random clones having moderate-to-high expression levels were scaled up to 50 mL scale expression and purified using standard immobilized metal affinity chromatography. Identities of all proteins were confirmed by mass spectrometry.

The random clones were analyzed for stability by DSC. Briefly, DSC measurements were conducted on a VP-Capillary DSC (MicroCal). Proteins were exchanged into PBS (pH 7.2) through extensive dialysis, and adjusted to a concentration of 0.25-0.5 mg/ml for DSC analysis. Samples were scanned from 20-95° C. at a scan rate of 90° C./hour, with no repeat scan. The results are shown in TABLE 18.

TABLE 18 Comparison of T_(m) values of Tn3s with FG9 vs FG10/11 FG9 T_(m)(° C.) FG10/11 T_(m)(° C.) A1 64.8 E12 (FG10) 65.0 A3 71.8 F5 (FG10) 60.0 B2 70.0 G1 (FG11) 64.3 B4 69.4 G4 (FG11) 67.6 C5 66.6 G8 (FG11) 64.2 C7 66.0 H6 (FG11) 70.3 C8 64.1 H7 (FG11) 71.7 C11 59.5 H8 (FG10) 61.9 D1 73.7 H9 (FG10) 59.5 D8 72.1 H10 (FG11) 67.6 D10 65.6 H11 (FG11) 63.7 D11 65.6 H12 (FG11) 65.6 D12 66.4 E1 75.0 E3 66.0 E9 75.3 E11 61.9 n = 17 n = 12 Mean 67.9 Mean 65.1

In this study, the thermal stability of Tn3s with loop length FG9 or FG10 and 11 was compared. The trend shows that Tn3 domains having an FG loop of length 9 are more thermostable than those with loop length FG10 or 11. A control, the wild-type Tn3 domain (with an FG loop of 10 residues) had a Tm of 72° C. when run in parallel with the above samples. The range of T_(m) values seen with each loop length indicates that other factors also play a role in determining Tn3 domain thermostability.

Example 12 Generation and Characterization of a Trispecific Tn3

In these experiments, a Tn3 molecule having binding specificity for three different targets was generated and characterized. D1, the Tn3 domain specific for the Synagis® antibody, was linked to 1E11, a Tn3 domain specific for TRAIL receptor 2, and 79, a Tn3 domain specific for CD40L, respectively (FIG. 17A). The construct was expressed in BL21(DE3) E. coli cells and purified using standard methods (see FIG. 17B).

To confirm that the trispecific constructs were capable of binding pairs of all three targets simultaneously, both AlphaScreen and ELISA experiments were conducted. For AlphaScreen experiments, trispecific Tn3, subsets of two of the three total target molecules (one biotinylated and the other containing an antibody Fc region), Protein A donor beads, and streptavidin acceptor beads were combined in a 384-well white Optiplate, as described above. AlphaScreen signal can only be observed when the streptavidin donor bead and Protein A acceptor bead are within proximity of each other (200 nm of each other), which in this assay is accomplished through bridging by the trispecific molecule. The ability of D1-1E11-79 to simultaneously bind huCD40L and TRAIL R2-Fc (FIG. 18A), and to simultaneously bind huCD40L and Synagis® (FIG. 18B) was confirmed by AlphaScreen as follows: in a 384-well white Optiplate, the following components were combined in a total volume of 30 μl: 20 mM purified D1-1E11-79, 50 mM biotinylated-huCD40L, (0, 1, 2.5, 5, 10, or 42 nM) TrailR2-Fc (FIG. 18A) or (0, 1, 2.5, 5, 10, or 42 nM) Synagis® (FIG. 18B), 5 μl each of 1/50 dilutions of AlphaScreen Protein A acceptor beads and streptavidin donor beads. After 1 hour incubation in the dark, the plate was read on an Envision plate reader in AlphaScreen mode.

Because Synagis® and TRAIL R2-Fc both contain an Fc domain, the AlphaScreen assay could not be used to demonstrate simultaneous binding of these molecules to the trispecific construct. In place of this, an ELISA experiment was conducted. MaxiSorp plates were coated with TRAIL R2-Fc (100 μl at 1 μg/ml), blocked with 4% milk, then followed by addition of varying concentrations of the trispecific construct. Biotinylated Synagis®, the second target ligand, was added and detected by the addition of HRP-streptavidin (FIG. 19). of D1-1E11-79 was also shown to be capable of binding both TRAIL R2-Fc and Synagis® simultaneously, as indicated by the ELISA results in FIG. 19. Therefore we can conclude that this construct can bind all three pairs of its targets simultaneously.

Example 13 Lead Isolation

The first step in developing an agonist Tn3 is to isolate a Tn3 monomer that can bind to TRAIL R2 and when linked into a multivalent format can bind two or more TRAIL R2 extracellular domains in a way that engages the apoptotic pathway. Since not all binders may act as agonists, we decided to first isolate a panel of binders and then screen for agonism in a secondary in vitro cell killing assay. We first panned a large phage displayed library of Tn3's with variation in the BC, DE, and FG loops on recombinant TRAIL R2-Fc to isolate an initial panel of binders. The Tn3 scaffold chosen as the basis for this library was not a native 3^(rd) FnIII domain from tenascin C but a version that had an engineered disulfide to improve stability. An in house Tn3/gene 3 fused phage display library was constructed containing randomization in the BC, DE, and FG loops. Multiple binders were found by a phage ELISA in which TRAIL R2 was directly coated on a plate and binding of 1:3 diluted phage in PBS+0.1% Tween 20 (PBST) 1% milk was detected by anti-M13-peroxidase conjugated antibody (GE Healthcare Biosciences, Piscataway, N.J.). A majority of the binders had an undesirable free cysteine in one of the loops and were not chosen for further study. A subset of the clones lacking an unpaired cysteine were cloned into expression vectors generating either an Fc fusion or antibody-like construct (FIG. 1) and tested in the tumor cell line H2122 for cell killing (data not shown). Although the Fc fusion format failed to kill cells regardless of its fused Tn3, the antibody-like format did elicit a response for more than one binder.

Example 14 Affinity Maturation

Clone 1C12 (SEQ ID NO: 132) (see FIG. 20) showed the best cell killing in the initial screening assays and was therefore chosen for affinity maturation. Affinity maturation was performed by saturation mutagenesis of portions of the loops using either Kunkel mutagenesis or PCR with oligonucleotides containing randomization, assembly, and ligation into the phage display vector. Round one and three consisted of saturation mutagenesis in parts of the BC and FG loops respectively and round 2 combined saturation mutagenesis of parts of all three loops separately, panning, gene shuffling, and then panning of the shuffled mutants to obtain the highest affinity output clone. Pools of affinity matured clones were recovered after panning by PCR directly from the phage or by prepping the single stranded DNA using a Qiagen kit (Qiagen, Valencia, Calif.) and then PCR. PCR products were digested NcoI to KpnI (New England Biolabs, Ipswich, Mass.) and cloned into our in house expression vector pSEC. The clones were expressed in MagicMedia (Invitrogen, Carlsbad, Calif.) and run on a gel to verify that expression did not differ greatly between clones. Improved clones were identified by a competition ELISA in which plates were coated with tetravalent, antibody-like 1C12 (SEQ ID NOs: 154 and 155), and the inhibition in binding of 0.75 nM TRAIL R2 biotin in the presence of dilutions of Tn3 in MagicMedia was measured using streptavidin-horseradish peroxidase (GE Healthcare Biosciences, Piscataway, N.J.). TMB (KPL, Gaithersburg, Md.) was added and neutralized with acid. Absorbance was read at 450 nm.

Affinity measurements were performed on the ProteOn XPR36 protein interaction array system (Bio-Rad, Hercules, Calif.) with GLC sensor chip at 25° C. ProteOn phosphate buffered saline with 0.005% Tween 20, pH 7.4 (PBS/Tween) was used as running buffer. TRAIL R2 was immobilized on the chip and a two-fold, 12 point serial dilution of the Tn3 binders (1C12 (SEQ ID NO: 132), 1E11 (SEQ ID NO: 134), G3 (SEQ ID NO: 133), C4 (SEQ ID NO: 135), and G6 (SEQ ID NO: 138)) were prepared in PBS/Tween/0.5 mg/ml BSA, pH 7.4 at starting concentrations ranging from 36 μM to 700 nM. Samples of each concentration were injected into the six analyte channels at a flow rate of 30 μl/min. for 300 seconds. The K_(d) was determined by using the equilibrium analysis setting within the ProteOn software. The sequences of the best clones from each round are shown in FIG. 20. The total improvement in affinity after three rounds of affinity maturation was almost two orders of magnitude with the best clones having affinities in the 40-50 nM range (TABLE 19).

TABLE 19 Equilibrium binding constants of monomeric best clones from affinity maturation of lead clone 1C12 as measured by Surface Plasmon Resonance (SPR). Round Clone K_(d)(nM) Fold Improvement Lead isolation 1C12 4130 ± 281 Affinity maturation 1 G3 422 ± 45 10 Affinity maturation 2 1E11 103 ± 9  40 Affinity maturation 3 C4 50 ± 2 83 Affinity maturation 3 G6 43 ± 2 96

Example 15 Effect of Tn3 Affinity on Potency in Antibody-Like Format

In order to assess the effect of affinity of the individual TN3 subunit on potency, all of the clones in TABLE 19 were reformatted into the antibody-like construct depicted in FIG. 1. To express the antibody-like proteins, 293F cells were transiently transfected with the appropriate expression constructs. Harvests of supernatant were performed on days 6 and 10 and the protein was purified by protein A affinity chromatography. All purified proteins were analyzed by SDS-PAGE on NuPage Novex 4-12% bis tris gels in MES buffer without reducing agent, and were visualized using SimplyBlue SafeStain (Invitrogen, Carlsbad, Calif.).

Size exclusion chromatography was also used to analyze purified proteins, and where necessary, aggregated material was removed on either a Superdex 75 10/300GL or Superdex 200 10/300GL column (GE Healthcare, Piscataway, N.J.), to a final level below 10% of total protein. An Acrodisc unit with a Mustang E membrane (Pall Corporation, Port Washington, N.Y.) was used as indicated by the manufacturer to remove endotoxin from bacterially expressed protein preparations.

H2122 cells were then tested for sensitivity to the agonistic antibody-like constructs using a CellTiter-Glo cell viability assay. In this assay, luminescence is directly proportional to the levels of ATP within a given well of a 96 well plate, which in turn is directly proportional to the amount of metabolically active viable cells. For the H2122 cell line, cells were plated in 96 well plates at a density of 10,000 cells/well in 75 μl of complete medium (RPMI 1640 medium supplemented with 10% FBS). Following overnight incubation at 37° C., media was supplemented with 25 μl of additional media containing a serial dilution of TRAIL R2-specific or negative control proteins. All treatments were performed in duplicate wells. Commercially available TRAIL ligand (Chemicon/Millipore, Billerica, Mass.) was used as a positive control for TRAIL receptor-induced cell death.

After 72 hours, the CellTiter-Glo kit was used according to the manufacturer's instructions. Assay luminescence was measured on an Envision Plate reader (PerkinElmer, Waltham, Mass.). Inhibition of cell viability was determined by dividing the luminescence values for treated cells by the average luminescence for untreated viable cells.

Two variables determine potency: the concentration at which a construct inhibits the viability of cells by 50% (EC₅₀) and the maximum inhibition of cell viability. FIG. 21 shows that as a general trend, greater affinity of the Tn3 monomer leads to a lower EC₅₀ of the antibody-like constructs as G6 has a lower EC₅₀ than 1E11 and 1E11 has a lower EC₅₀ than 1C12.

Example 16 Pharmacokinetics of Linear Tn3's

To determine the half life of the linear Tn3 tandems as a function of the number of Tn3 modules per tandem, the G6 monomer (SEQ ID NO: 138), G6 tandem 4 (SEQ ID NO: 143), G6 tandem 6 (SEQ ID NO: 192), and G6 tandem 8 (SEQ ID NO: 145) were injected into a mouse and serum concentration of the Tn3s was monitored by an ELISA. The route of administration was intraperitoneal (IP) injection. The experimental design is shown in TABLE 20. Mice were bled 150 μl per time point. Tn3's were detected in serum by an ELISA in which in house produced TRAIL R2 coated plates were incubated with serum diluted in PBST 1% milk. Initial ELISAs were performed to determine for a given time point the correct dilution range in order for the signal to be within the dynamic range of the assay. Bound Tn3 was detected with a 1 in 1,000 dilution of polyclonal anti-Tn3 serum from rabbit in PBST 1% milk (Covance, Princeton, N.J.) followed by a 1 in 10,000 dilution in PBST 1% milk of donkey anti-rabbit HRP (Jackson ImmunoResearch, West Grove, Pa.). For each construct, a standard curve was made. Statistical analysis was performed using an in house statistical program.

The term “maximum plasma concentration” (“C_(max)”) refers to the highest observed concentration of tandem Tn3 in plasma following administration of the test material to the patient.

The term “T_(max)” refers to the time to maximum plasma concentration C_(max).

The term “area under the curve” (“AUC”) is the area under the curve in a plot of the concentration of tandem Tn3 in plasma against time. AUC can be a measure of the integral of the instantaneous plasma concentrations (C_(p)) during a time interval and has the units of mass*time/volume. However, AUC is usually given for the time interval zero to infinity. Thus, as used herein “AUC_(inf)” refers to an AUC from over an infinite time period.

The term “biological half-life” (“T_(1/2)”) is defined as the time required for the plasmatic concentration of tandem Tn3 to reach half of its original value.

The term “CL/F” refers to the apparent total body clearance calculated as Dose/AUC_(inf).

Tn3 biological half-life (T_(1/2)) increases with increasing number of tandem Tn3's per linear molecule. Adding seven Tn3's to make a tandem 8 from a monomer increased the half life by almost 50%. Increases in valency did not affect the T. However, increases in valency from 1 to 8 resulted in approximately ten-fold and 7-fold increases in C_(max) and AUC_(inf), respectively. Furthermore, when valency increase from 1 to 8, an approximately 7-fold decrease in clearance (CL/F) was observed.

TABLE 20 Experimental design of anti-TRAIL R2 linear tandem pharmacokinetic assay. Group # Test Dose Route Volume Time points # Mice 1 G6 10 mg/kg IP 10 ml/kg (15 min, 1 hr, 16 hr), (3) (3) (3) monomer (30 min, 4 hr, 24 hr) (2 hr, 6 hr, 48 hr) 2 G6 10 mg/kg IP 10 ml/kg (15 min, 1 hr, 16 hr), (3) (3) (3) tandem 4 (30 min, 4 hr, 24 hr) (2 hr, 6 hr, 48 hr) 3 G6 10 mg/kg IP 10 ml/kg (15 min, 1 hr, 16 hr), (3) (3) (3) tandem 6 (30 min, 4 hr, 24 hr) (2 hr, 6 hr, 48 hr) 4 G6 10 mg/kg IP 10 ml/kg (15 min, 1 hr, 16 hr), (3) (3) (3) tandem 8 (30 min, 4 hr, 24 hr) (2 hr, 6 hr, 48 hr) Total 36

TABLE 21 Pharmacokinetic properties of Tandem Tn3's Pharmacokinetic Parameters Test C_(max) T_(max) AUC_(inf) T_(1/2) CL/F Material (μg/mL) (hr) (hr · μg/mL) (hr) (mL/hr/kg) G6 monomer 3.65 1 9.31 1.22 1070 G6 tandem 4 8.07 1 23.2 1.46 431 G6 tandem 6 24.6 1 36.5 1.69 274 G6 tandem 8 38.6 1 64.2 1.76 156

Example 17 Engineered Enhancement of Cyno Cross-reactivity

For pre-clinical toxicity testing in cynomolgus monkeys (Macaca fascicularis), it is desirable to develop an anti-TRAIL R2-Tn3 that cross reacts with cynomolgus TRAIL R2 (cyno TRAIL R2). Our initial affinity matured lead clones had poor cross reactivity with cyno TRAIL R2, although the homology to human TRAIL R2 is 88%. The cross reactivity was enhanced by making a library based upon clone F4 (SEQ ID NO: 137), which was the clone with the best cyno cross reactivity among the clones that resulted from affinity maturation.

Two libraries were made by saturation mutagenesis: one with diversity in the FG loop alone and one with diversity in the BC and FG loops. A low error rate mutagenic PCR was also used to allow for mutations outside the loops that may be beneficial for enhanced cyno TRAIL R2 binding. Four rounds of phage panning were done on in house produced cyno TRAIL R2, and outputs were cloned into the pSEC expression vector. For screening of initial hits in an ELISA format, Tn3's were secreted into MagicMedia (Invitrogen, Carlsbad, Calif.) and were captured from supernatant using an anti-his tag antibody (R and D Systems, Minneapolis, Minn.).

Binding of either human or cyno TRAIL R2-Fc in solution to captured Tn3 was detected by anti-human-Fc-HRP. Clones that had significant binding to cyno TRAIL R2-Fc and did not appear to lose binding to human TRAIL R2-Fc were selected for a subsequent screening ELISA in which either human or cyno TRAIL R2-Fc was coated on a plate and Tn3 supernatants were titrated and then detected with anti-his tag HRP. Because the level of variation in expression levels from clone to clone was low, and also because avidity from having divalent TRAIL R2-Fc in solution could not mask differences in Tn3 affinity, this ELISA allowed for affinity discrimination. It was found that one mutation, a mutation from D to G two amino acids before the DE loop, was present in all engineered cyno cross reactive clones (FIG. 23A). This D to G mutation was engineered into the original F4 to make a clone named F4 mod 1 (SEQ ID NO: 193) and the cross reactivity for cyno was greatly improved without sacrificing binding to human TRAIL R2 (FIG. 23B). In this ELISA, inhibition of binding of 0.75 nM of human or cyno TRAIL R2-Fc to F4 mod 1 coated plates by purified F4 or F4 mod 1 was measured.

It is desired that the binding of a cyno cross reactive enhanced clone to cyno TRAIL-R2-Fc be within tenfold of its binding to human TRAIL R2-Fc. Also, it is desired that the binding of a cyno cross reactive enhanced clone to cyno TRAIL-R2-Fc be within tenfold of the binding of F4 to human TRAIL R2-Fc. The IC₅₀ for F4 mod 1 binding to cyno TRAIL R2 differs by less than three fold from the IC₅₀ for F4 mod 1 binding to human TRAIL R2. In addition, the IC₅₀ for F4 mod 1 binding to human TRAIL R2 is six-fold stronger than the IC₅₀ for F4 binding to human TRAIL R2. Accordingly, F4 mod 1 meets the intended cross reactivity requirements.

Example 18 Germline Engineering of Enhanced Cyno Cross Reactive Clone

Clone F4 mod 1 was further engineered to eliminate non essential mutations from germline in order to reduce possible immunogenicity risk. A panel of twelve different modifications was made to determine if there was an effect from a given mutation on the binding to both human and cyno TRAIL R2. FIG. 24A shows a comparison of the final clone F4 mod 12 (SEQ ID NO: 194), which incorporates all tested germline mutations that do not affect binding, to other constructs, namely the Tn3 germline, the original F4 parent, and clone F4 mod 1 (initial enhanced cyno cross reactive engineered).

The amino acid sequence of F4 mod 12 starts with the native Tn3 sequence SQ, ends with L, has a reversion of the framework 2 mutation from A to T, and has a reversion of the final two amino acids of the DE loop from TA to NQ. FIG. 24B shows that F4, F4 mod 1, and F4 mod 12 all are within six-fold of each other in their binding to human TRAIL R2. It also shows that F4 mod 1 and F4 mod 12 are within twofold of each other in their binding to cyno TRAIL R2.

F4 mod 12 was reformatted into a tandem 6 (SEQ ID NO: 167) and tandem 8 (SEQ ID NO: 166) construct and tested to confirm that there is not loss in potency relative to G6 tandem 6 (SEQ ID NO: 144) and tandem 8 (SEQ ID NO: 145). FIG. 24C and FIG. 24D show no loss in potency for the germline engineered, enhanced cyno cross reactive F4 mod 12 tandems in comparison to the G6 tandems in the Colo205 cell line.

Example 19 Activity of G6 Tandem 8 in TRAIL Resistant Cell Lines

Multiple cell lines are resistant to killing by TRAIL. Thus, we evaluated whether the enhanced potency of G6 tandem 8 constructs relative to TRAIL in TRAIL sensitive cell lines will translate into potency of G6 tandem 8 in TRAIL resistant cell lines. Sensitivity to Apo2L/TRAIL in several cancer cell lines was determined with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, Wis.). Briefly, cells were plated in 96-well plates, allowed to adhere overnight and then treated with various concentrations of recombinant human Apo2L/TRAIL and TRAIL mimetic G6 Tandem 8 in medium containing 10% FBS. After a period of 48-72 hrs, cell viability was determined following manufacturer's protocols. FIG. 25 shows that for the TRAIL resistant cell line HT29 G6 tandem 8 shows potent cell killing activity while TRAIL does not. TABLE 22 shows that G6 tandem 8 has cell killing activity in many, but not all of the TRAIL resistant cell lines tested.

TABLE 22 Activity of G6 tandem 8 and TRAIL in TRAIL resistant cell lines. G6 TRAIL G6 TRAIL Tandem 8 % Max Tandem 8 IC50 [nM] IC50 [nM] Kill % Max Kill Resistant T84 >8.3 0.247 14.44 71.53 to TRAIL LoVo >8.3 0.005 45.99 74.22 but CaCo-2 >8.3 0.044 18.23 54.84 sensitive HT29 >8.3 0.01 28.00 85.40 to TRAIL HPAF-II >8.3 0.0

6 45.33 91.33 mimetics Hep3B >8.3 0.023 13.35 70.15 SKHEP-1 >8.3 0.055 19.48 80.19 HepG2 >8.3 0.040 33.31 84.00 Resistant SW620 >8.3 >10 −5.71 4.65 to TRAIL SW837 >8.3 >10 19.98 25.32 and Hs766T >8.3 >10 20.99 47.86 TRAIL NCI-H522 >8.3 >10 32.69 31.38 mimetics

CI-H23 >8.3 >10 22.08 39.59 BT-549 >8.3 >10 4.49 27.99 SNB-7

>8.3 >10 8.9 4.7 786-0 >8.3 >10 −0.12 7.19 SN

-387 >8.3 >10 −0.63 33.1 SN

-475 >8.3 >10 0.49 20.88 SN

-393 >8.3 >10 1.50 0.46

indicates data missing or illegible when filed

Example 20 Immunogenicity Study of TRAIL R2 Binding Monomers

Immunogenicity is a potential issue for any therapeutic protein even if it is human in origin. Immunogenic responses can limit efficacy through neutralizing antibodies that can lead to inflammation. One of the most important factors in the development of an immune response is the presence of epitopes that can stimulate CD4+ T cell proliferation. In the EpiScreen test (Antitope, Cambridge, UK), CD8+ T cell depleted Peripheral Blood Mononuclear Cells (PBMCs) are incubated with test proteins and CD4+ T cell proliferation and IL-2 secretion are monitored (see, Baker & Jones, Curr. Opin. Drug Discovery Dev. 10:219-227, 2007; Jaber & Baker, J. Pharma. Biomed. Anal. 43:1256-1261, 2007; Jones et al., J. Thrombosis and Haemostasis 3:991-1000, 2005; Jones et al., J. Interferon Cytokine Res. 24:560-72, 2004). The PBMCs are isolated from a pool of donors which represent the HLA-DR allotypes expressed in the world's population.

The Tn3 monomers shown in FIG. 26 were expressed (with a GGGGHHHHHHHH linker-His tag), purified, and verified to be monomeric by SEC, and filtered for endotoxin removal as described above. All non-wild type clones tested were from the engineering round to enhance cyno cross reactivity (FIG. 23A). However, these clones had mutations to germline that have been shown not to affect binding in the F4 mod 1 background. These clones were tested in an ELISA to verify that the germlining mutations did not affect binding. In both the T cell proliferation assay and the IL-2 secretion assay, a stimulation index (SI) of greater than two had been previously established as a positive response for a given donor. The mean SI, or average of the SI of the positive responding population, is indicative of the strength of the response. A control protein known to induce a strong response, keyhole limpet haemocyanin (KLH), was included in both assays.

TABLE 23 shows the mean SI for all test proteins, which was significantly lower than for KLH and was not much higher than the cutoff of 2 for a positive mean SI. In addition, the frequency of response for the test proteins was very low (ten percent or less for all tested proteins except for the control which had a response in excess of 90%). Previous studies by Antitope have revealed that an EpiScreen response of less than 10% is indicative of low clinical immunogenicity risk. Thus, our observation that all Tn3s tested have 10% or less frequency of response indicates a low risk of clinical immunogenicity.

TABLE 23 Results of Antitope EpiScreen immunogenicity assay. Tested Tn3s are ranked from 1 (most immunogenic) to 4 (least immunogenic). Frequency (%) Mean SI of Response Sample Prolif IL-2 Prolif IL-2 Ranking F4mod12 2.82 2.30 4 4 4= 00322S-A07 2.91 2.06 8 8 2  00322S-G09 2.88 2.26 10 10 1  00322V-A10 2.67 2.33 8 6 3= 00322V-F11 3.14 2.37 6 6 3= wild type 2.05 2.00 6 4 4= KLH 6.51 3.98 96 92 N/A

Example 21 Aggregation State of Unpurified and Purified G6 Tandem 8 Tn3's

It is known in the art that proteins containing multiple cysteines, e.g., a protein made up of tandem repeats that contains an internal disulfide bond, often does not exhibit proper disulfide pairing. Scrambling of disulfides can reduce or eliminate expression into media. If the protein does express into media, it may be a mixture of improperly folded protein with intermolecular as well as mismatched intramolecular disulfide pairs leading to aggregation. Our SEC data revealed that the majority of the tandem proteins in the bacterial expression media were in a monomeric, properly folded state. After Ni-NTA purification of the Hi-tagged G6 tandem 8 protein, approximately 15% of the protein was aggregated. The observed aggregation was reduced to 4% (FIG. 27A) by reduction with 2 mM DTT, indicating that most of the aggregation was disulfide mediated. Most of the aggregates were removed by SEC purification (FIG. 27B), as described above.

Example 22 Determination of TRAIL Mimetics, G6TN6 and G6TN8, Tumor Growth Inhibition of in Colo205 Colorectal Cancer Xenograft Models

The anti-tumor activity of TRAIL Tn3 mimetics, G6 tandem 6 (G6TN6) (SEQ ID NO: 144) and G6 tandem 8 (G6TN8) (SEQ ID NO: 145), were evaluated in Colo205, a human colorectal carcinoma xenograft model. Colo205 cells were maintained as a semiadhesive monolayer culture at 37° C. under 5% CO₂ in Roswell Park Memorial Institute (RPMI) 1640 medium that contained 10% fetal bovine serum (FBS). Cells harvested by trypsinization were resuspended to a final concentration of 3×10⁷ cells/mL in Hank's balanced salt solution (HBSS). Athymic female nude mice were each injected subcutaneously (SC) in the right flank with 3×10⁶ Colo205 cells. The study was initiated when tumors reached an average of ˜177 mm³. The study design is summarized in TABLE 24. TRAIL was diluted from stock solution with 20 mM Tris-HCl 300 mM Arginine-HCl pH 7 and administered intravenously (IV) at dose indicated in TABLE 24, daily for a total of 5 doses according to body weight (10 mL/kg). G6 tandem 6 (G6TN6) and G6 tandem 8 (G6TN8) were each diluted from a stock solution with PBS and administered intravenously (IV) at doses indicated in TABLE 24, daily for a total of 5 doses according to body weight (10 mL/kg). Tumor volumes and body weight measurements were recorded. Tumor measurements were made using an electronic caliper and tumor volume (mm³) was calculated using the formula tumor volume=[length (mm)×width (mm)²]/2. Tumor growth inhibition (TGI) was calculated as percent TGI=(1−T/C)×100, where T=final tumor volumes from a treated group after the last dose, and C=final tumor volumes from the control group after the last dose.

During the dosing phase (DP) (FIG. 28), 3 mg/kg and 30 mg/kg of G6TN6 resulted in significant TGI of 92% (p<0.0001) and 93% (p<0.0001), respectively (TABLE 25). Similarly, after equimolar adjustment for final concentration, 2.25 mg/kg and 25.5 mg/kg of G6TN8 resulted in significant TGI of 93% (p<0.0001) and 94% (p<0.0001), respectively (TABLE 25). 30 mg/kg of TRAIL resulted in TGI of 60% (p<0.001), (TABLE 25).

By day 34 of the regrowth phase (RP) (FIG. 28, while 3 mg/kg G6TN6 did not result in any CR (complete regression), 2.25 mg/kg G6TN8 resulted in a 90% CR. At a higher dose of 30 mg/kg G6TN6 50% CR was achieved. On the other hand, 25.5 mg/kg G6TN8 resulted in 100% CR (TABLE 26). Results from both doses suggest that G6TN8 resulted in greater efficacy in comparison to G6TN6. However, both showed efficacy at certain doses. More importantly, both constructs significantly outperformed TRAIL which did not result in any PR or CR.

As shown in FIG. 29, no body weight loss was observed for both G6TN6 and G6TN8 at all doses during the dosing and regrowth phase of the study.

TABLE 24 Study design for Trail and TRAIL mimetics (G6TN6 and G6TN8) in Colo205 tumor xenograft model Dose Test Dose Volume Dose Group Material (mg/kg) (mL/kg) Route Schedule 1 Untreated NA NA NA NA 2 PBS NA 10 IV QDX5 3 Trail   30 mg/kg 10 IV QDX5 4 G6TN6   30 mg/kg 10 IV QDX5 5 G6TN6   3 mg/kg 10 IV QDX5 6 G6TN8 25.5 mg/kg 10 IV QDX5 7 G6TN8 2.25 mg/kg 10 IV QDX5

TABLE 25 Effect of TRAIL and TRAIL mimetics (G6TN6 and G6TN8) on TGI during dosing phase of the study. P Value (compared to Treatment group % TGI untreated control) Trail 30 mg/kg 60 P < 0.001  G6TN6 30 mg/kg 93 P < 0.0001 G6TN6 3 mg/kg 92 P < 0.0001 G6TN8 25.5 mg/kg 94 P < 0.0001 G6TN8 2.25 mg/kg 93 P < 0.0001

TABLE 26 Effect of TRAIL and TRAIL mimetics (G6TN6 and G6TN8) on TGI during regrowth phase by day 34 of the study. Treatment group PR^(a) (%) CR^(b) (%) Trail — — G6TN6 30 mg/kg 50 50 G6TN6 3 mg/kg 100  — G6TN8 25.5 mg/kg — 100  G6TN8 2.25 mg/kg 10 90 ^(a)percent partial regression (PR; percentage of mice in group where tumor volume is less than 50% of volume at time of staging for two successive measurements) ^(b)percent complete regression (CR; percentage of mice in group where no palpable tumor detectable for two successive measurements)

Example 23 Binding Additional Targets

FnIII scaffolds that bind to particular targets may be generated by the methods described herein and/or known in the art (see for Example WO 2009/058379). Alternatively, the scaffolds described herein are subjected to “loop grafting” in which the loop sequences of a scaffold of known binding specificity are grafted to the beta strand sequences of the desired scaffold (e.g., the beta strand sequences of a Tn3 scaffold or the sequences presented in FIG. 16). TABLE 27 provides a non-limiting example of loop sequences for grafting to desired beta strands, for example those provided in TABLE 1.

TABLE 27 Loop Sequences for Loop Grafting AB Loop BC Loop CD Loop DE Loop EF Loop FG Loop Target (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) αvβ3 Integrin VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTLRGDWSEDSKPI See: US7,7556,925 (NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 210) VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTVRGDWYEYSKPI (NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 211) VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTGRGDWTEHSKPI (NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 212) VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTARGDWVEGSKPI (NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 213) VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTPRGDWTEGSKPI (NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 214) VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTPRGDWIEFSKPI (NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 215 VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTGRGDWNEGSKPI (NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 216) VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTFRGDWIELSKPI (NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 217) Estrogen Receptor VAATPWTWV DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTGRGDSPASSKPI See: US7,598,352 LRETS (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 60) (NO: 218) VAATPWVLI DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTGRGDSPASSKPI TRSTS (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 60) (NO: 219) VAATPTS DAPWYQGRY TGGNSPV SGLKPGVD SGLKPGVD VTGRLRAQLVSKPI (NO: 55) (NO: 220) (NO: 57) (NO: 58) (NO: 59) (NO: 221) VAATPTS DAPRTKQY TGGNSPV SGLKPGVD SGLKPGVD VTGRLRDLLQSKPI (NO: 55) (NO: 222) (NO: 57) (NO: 58) (NO: 59) (NO: 223) VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VRGLVRFRVVNSSL (NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) CMWARSKPI (NO: 224) VEGFR-2 VAATPTS RHPHFPTRY TGGNSPV PLQPPL SGLKPGVD VTKERNGRELFTPI See: US7858739 (NO: 55) (NO: 225) (NO: 57) (NO: 226) (NO: 59) (NO: 227) VAATPTS RHPHFPTRY TGGNSPV PLQPPT SGLKPGVD VTDGRNGRLLSIPI (NO: 55) (NO: 225) (NO: 57) (NO: 228) (NO: 59) (NO: 229) VAATPTS RHPHFPTRY TGGNSPV PLQPPT SGLKPGVD VTMGLYGHELLTPP (NO: 55) (NO: 225) (NO: 57) (NO: 228) (NO: 59) I (NO: 230) VAATPTS RHPHFPTRY TGGNSPV PLQPPT SGLKPGVD VTDGENGQFLLVPI (NO: 55) (NO: 225) (NO: 57) (NO: 228) (NO: 59) (NO: 231) EGFR VAATPTS HERDGSRQY TGGNSPV PGGVRT SGLKPGVD VTDYFNPTTHEYIY See: WO2010/060095 (NO: 55) (NO: 232) (NO: 57) (NO: 233) (NO: 59) QTTPI (NO: 234) VAATPTS WAPVDRYQY TGGNSPV PRDVYT SGLKPGVD VTDYKPHADGPHTY (NO: 55) (NO: 235) (NO: 57) (NO: 236) (NO: 59) HESPI (NO: 237) VAATPTS TQGSTHYQY TGGNSPV PGMVYT SGLKPGVD VTDYFDRSTHEYKY (NO: 55) (NO: 238) (NO: 57) (NO: 239) (NO: 59) RTTPI (NO: 240) VAATPTS YWEGLPYQY TGGNSPV PRDVNT SGLKPGVD VTDWYNPDTHEYIY (NO: 55) (NO: 241) (NO: 57) (NO: 242) (NO: 59) HTIPI (NO: 243) IGF-IR VAATPTS SPYLRVARY TGGNSPV PSSART SGLKPGVD VTPSNIIGRHYGPI See: WO2008/066752 (NO: 55) (NO: 244) (NO: 57) (NO: 245) (NO: 59) (NO: 246) VAATPTS VNDPQRNRY TGGNSPV PAYYPT SGLKPGVD VTYSHIKYLYHKPI (NO: 55) (NO: 247) (NO: 57) (NO: 248) (NO: 59) (NO: 249) VAATPTS SDSLKVSRY TGGNSPV PKQYHT SGLKPGVD VTPSNIIGRHYGPI (NO: 55) (NO: 250) (NO: 57) (NO: 251) (NO: 59) (NO: 252) VAATPTS SAPLKVARY TGGNSPV PKNVYT SGLKPGVD VTKMRDYRPI (NO: 55) (NO: 253) (NO: 57) (NO: 254) (NO: 59) (NO: 255)

The foregoing examples illustrate various aspects of the invention and practice of the methods of the invention. The examples are not intended to provide an exhaustive description of the many different embodiments of the invention. Thus, although the forgoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill in the art will realize readily that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. 

1-76. (canceled)
 77. A recombinant multimeric scaffold comprising two fibronectin type III (FnIII) monomer scaffolds derived from one or more FnIII domains of interest (FOI), wherein (a) each FnIII monomer scaffold comprises seven beta strands designated A, B, C, D, E, F, and G linked to six loop regions designated AB, BC, CD, DE, EF, and FG, (b) the FnIII monomer scaffolds are connected in tandem, wherein at least two of the monomers comprises a non-naturally occurring intramolecular disulfide bond, (c) the recombinant multimeric scaffold specifically binds to at least one target, and (d) the action on the target is improved over that of a cognate FnIII monomer scaffold.
 78. The multimeric scaffold of claim 77, wherein the multimeric scaffold comprises 3, 4, 5, 6, 7, or 8 FnIII monomer scaffolds.
 79. The multimeric scaffold of claim 77, wherein at least two FnIII monomer scaffolds are connected by a linker.
 80. The multimeric scaffold of claim 77, wherein at least to FnIII monomer scaffolds are directly connected without a linker interposed between the FnIII monomer scaffolds.
 81. The multimeric scaffold of claim 77, wherein at least one of the FnIII monomer scaffolds is fused to a heterologous moiety.
 82. The multimeric scaffold of claim 77, wherein at least two FnIII monomer scaffolds are different.
 83. The multimeric scaffold of claim 77, wherein each beta strand has at least 50% homology to the cognate beta strand of a FnIII domain of interest (FOI) and at least one loop is a non-naturally occurring variant of the cognate loop in the FOI.
 84. The multimeric scaffold of claim 77, wherein at the FOI of comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 1-4 and
 14. 85. The multimeric scaffold of claim 77, wherein the beta strands of at least one of the FnIII monomer scaffolds have at least 90% sequence identity to the cognate beta strands in SEQ ID NO: 1, 4 or
 14. 86. The multimeric scaffold of claim 77, wherein for at least one FnIII monomer scaffold, the A beta strand comprises SEQ ID NO: 41 or 42, the B beta strand comprises SEQ ID NO: 43, the C beta strand comprises SEQ ID NO: 45, or 131, the D beta strand comprises SEQ ID NO: 46, the E beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO: 49, 50 or 51, and the G beta strand comprises SEQ ID NO: 52 or
 53. 87. An isolated nucleic acid molecule encoding the multimeric scaffold of claim
 77. 88. A composition comprising the recombinant multimeric scaffold of claim 77 in a pharmaceutically acceptable excipient.
 89. A multimeric scaffold comprising two fibronectin type III (FnIII) monomer scaffolds, wherein each FnIII scaffold binds a target and wherein at least one FnIII monomer scaffold comprises the amino acid sequence: IEV(X_(AB))_(n)ALITW(X_(BC))_(n)CELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n) YEVSLIC(X_(FG))_(n)FETFTT, or IEV(X_(AB))_(n)ALITW(X_(BC))_(n)IELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n) YCVSLIS(X_(FG))_(n)KECFTT, or IEV(X_(AB))_(n)ALITW(X_(BC))_(n)CELX₁YGI(X_(CD))_(n)TTIDL(X_(DE))_(n)YSI(X_(EF))_(n) YCVSLIC(X_(FG))_(n)KECFTT

wherein X_(AB), X_(BC), X_(CD), X_(DE), X_(EF), and X_(FG) represent the amino acid residues present in the AB, BC, CD, DE, EF, and FG loops, respectively, wherein X₁ represents amino acid residue A or T, and wherein the length of the loop n=2-26
 90. The multimeric scaffold of claim 89, wherein the multimeric scaffold comprises 3, 4, 5, 6, 7, or 8 FnIII monomer scaffolds.
 91. The multimeric scaffold of claim 89, wherein at least two FnIII monomer scaffolds are connected by a linker.
 92. The multimeric scaffold of claim 89, wherein at least to FnIII monomer scaffolds are directly connected without a linker interposed between the FnIII monomer scaffolds.
 93. The multimeric scaffold of claim 89, wherein at least two FnIII monomer scaffolds are different.
 94. The multimeric scaffold of claim 89, wherein the AB loop comprises SEQ ID NO: 35, the CD loop comprises SEQ ID NO: 37, and the EF loop comprises SEQ ID NO: 39
 95. The multimeric scaffold of claim 89, wherein the BC loop comprises SEQ ID NO: 36, the DE loop comprises SEQ ID NO: 38, and the FG loop comprises SEQ ID NO:
 40. 96. An isolated nucleic acid molecule encoding the multimeric scaffold of claim
 89. 97. A composition comprising the recombinant multimeric scaffold of claim 89 in a pharmaceutically acceptable excipient. 